Corneal endothelium ecm therapeutic medicaments

ABSTRACT

The present invention provides medicaments for treating or preventing a disease, disorder, or condition associated with extracellular matrix (ECM) abnormality in a corneal endothelium, wherein the medicaments comprise a TGF-beta signal inhibiting agent. More specifically, this disease, disorder, or condition is a disorder associated with Fuchs&#39; endothelial corneal dystrophy. Such a disorder includes photophobia, blurred vision, vision disorder, eye pain, lacrimation, hyperemia, pain, bullous keratopathy, ophthalmic unpleasantness, a decrease in contrast, glare, edema in corneal stroma, bullous keratopathy, corneal opacity, and the like. A preferable TGF-beta signal inhibiting agent includes 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending U.S. patentapplication Ser. No. 14/907,881, filed Jan. 27, 2016, which is the U.S.national phase of International Patent Application No.PCT/JP2013/071095, filed Jul. 30, 2013. The contents of the foregoingapplications are hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention is related to techniques, methods, agents, and thelike to treat or prevent a disease, disorder, or condition associatedwith extracellular matrix (ECM) abnormality in a corneal endothelium.

BACKGROUND ART

Visual information is recognized when light transmitted into the cornea,which is a transparent tissue at the front-most part of an eye ball,reaches the retina and excites nerve cells of the retina and a generatedelectric signal is transmitted through the optic nerve to the visualcortex of the cerebrum. To attain good vision, it is necessary that thecornea is transparent. The transparency of the cornea is maintained bymaintaining constant water content with pumping and barrier functions ofcorneal endothelial cells.

Human corneal endothelial cells are present at a density of about 3000per 1 mm² at birth. However, once damaged, the ability of the cells toregenerate is highly limited. Fuchs' endothelial corneal dystrophy is adisease in which an abnormality occurs in the endothelial cells insideof the cornea to cause edema of the cornea and the like. The causethereof is unknown. In Fuchs' endothelial corneal dystrophy, anextracellular matrix, such as collagen and the like, deposits on onepart of the back surface of Descemet's membrane at the back side of thecornea to cause corneal guttae and thickening of Descemet's membrane.Corneal guttae and thickening of Descemet's membrane are causes ofphotophobia and blurred vision in Fuchs' endothelial corneal dystrophypatients and significantly impair the QOL of the patients. For Fuchs'endothelial corneal dystrophy, there is considered no effectivetherapeutic method other than keratoplasty. However, cornea donation isinsufficient in Japan. While there are about 2600 patients waiting forkeratoplasty, the number of keratoplasty cases performed domestically isabout 1700 annually.

With regard to Fuchs' endothelial corneal dystrophy, culture of cornealendothelial cells derived from Fuchs' endothelial corneal dystrophypatients (Non Patent Literatures 1 and 3) as well as immobilizationthereof (Non Patent Literatures 2) were reported, while a suitable cellfor screening for a therapeutic medicament and an advance-preventingmedicament that keeps characteristics of the disease, such asaccompanying overproduction of a extracellular matrix, is not reported.Accordingly, there is a limit on development of the therapeuticmedicament. Currently, there is no therapeutic medicament clinicallyused, and keratoplasty has to be relied on.

In addition, Patent Literature 1 discloses a TGF-beta1 inhibitor peptideto perform therapy against corneal fibrosis and/or opacity. PatentLiterature 2 discloses antibodies that bind to TGF-beta1, 2, or 3.Patent Literature 3 discloses that an Nrf2 agonist or activator may beused in therapy against corneal endothelial disorder. Patent Literature4 discloses a peptide that can bind to a transforming growth factorTGF-beta1 and become a strongly inhibiting agent against the bioactivityof TGF-beta1 by direct binding with a cytokine. Patent Literature 5discloses a scar-formation suppressing agent including BMP-7polypeptide. Patent Literature 6 generally describes a corneal disorderas a disease against which the TGF-beta inhibitory effect istherapeutically or preventively effective.

CITATION LIST Patent Literature

-   [PTL 1] Japanese National Phase PCT Laid-Open Publication No.    2013-520405-   [PTL 2] International Publication No. WO 2012/167143 pamphlet-   [PTL 3] International Publication No. WO 2012/009171 pamphlet-   [PTL 4] Japanese National Phase PCT Laid-Open Publication No.    2007-525204-   [PTL 5] Japanese National Phase PCT Laid-Open Publication No.    2006-508169-   [PTL 6] International Publication No. WO 2004/018430 pamphlet

Non Patent Literature

-   [NPL 1] Zaniolo K, et al., Exp. Eye Res. Vol. 94(1):22-31, 2012.-   [NPL 2] Azizi B, et al., Invest Ophthalmol Vis. Sci. 2;    52(13):9291-9297, 2011.-   [NPL 3] Kelliher C. et al., Exp. Eye Res. Vol. 93(6), 880-888, 2011.

SUMMARY OF INVENTION Solution to Problem

The inventors have found that the inhibition of a transforming growthfactor-beta (TGF-beta) pathway can suppress the deposition of anextracellular matrix (ECM) such as collagen and the like, as seen inFuchs' endothelial corneal dystrophy and the like; have found techniquesthat can treat or prevent a ECM-associated disorder; and have completedthe present invention. Accordingly, the invention of the presentapplication provides those inventions as follows:

-   -   (1) A medicament for treating or preventing a disease, disorder,        or condition associated with extracellular matrix (ECM)        abnormality in a corneal endothelium, wherein the medicament        comprises a TGF-beta signal inhibiting agent.    -   (2) The medicament for treating or preventing according to item        1, wherein the disease, disorder, or condition is a disorder        related to Fuchs' endothelial corneal dystrophy.    -   (3) The medicament for treating or preventing according to item        1 or 2, wherein the disease, disorder, or condition comprises at        least one selected from the group consisting of photophobia,        blurred vision, vision disorder, eye pain, lacrimation,        hyperemia, pain, bullous keratopathy, ophthalmic unpleasantness,        a decrease in contrast, glare, edema in corneal stroma, bullous        keratopathy, and corneal opacity in Fuchs' endothelial corneal        dystrophy.    -   (4) The medicament for treating or preventing according to any        one of items 1 to 3, wherein the TGF-beta signal inhibiting        agent comprises at least one of        4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide,        BMP-7, anti-TGF-beta antibody, anti-TGF-beta receptor antibody,        siRNA of TGF-beta, siRNA of a TGF-beta receptor, shRNA of        TGF-beta, shRNA of a TGF-beta receptor, an aptamer of TGF-beta,        an aptamer of a TGF-beta receptor, an antisense oligonucleotide        of TGF-beta,        6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone,        3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide,        2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,        6-(4-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine,        2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine,        4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, pharmaceutically        acceptable salts or solvates thereof, or solvates of the        pharmaceutically acceptable salts.    -   (5) The medicament for treating or preventing according to any        one of items 1 to 4, wherein the TGF-beta signal inhibiting        agent comprises        4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide        or a pharmaceutically acceptable salt thereof.    -   (6) The medicament for treating or preventing according to any        one of items 1 to 5, wherein the corneal endothelium is of a        primate.    -   (7) The medicament for treating or preventing according to any        one of items 1 to 6, wherein the corneal endothelium is of a        human.    -   (8) The medicament for treating or preventing according to any        one of items 1 to 7, comprising an additional medicinal        component.    -   (9) The medicament for treating or preventing according to any        one of items 1 to 8, being eye-drops.    -   (10) A TGF-beta signal inhibitory substance for treatment or        prevention of a disorder associated with extracellular matrix        (ECM) abnormality in a corneal endothelium.    -   (10A) The TGF-beta signal inhibitory substance according to        (10), wherein the TGF-beta signal inhibitory substance has a        characteristic of the inhibiting agent according to any one        of (1) to (9).    -   (11) A method for treating or preventing a disorder associated        with extracellular matrix (ECM) abnormality in a corneal        endothelium in a subject, wherein the method comprises a step of        administering an effective amount of a TGF-beta signal        inhibiting agent to the subject.

In the present invention, in addition to the clarified combinations, theabove-mentioned one or more characteristics are intended as beingfurther combined and provided. Still further embodiments and advantagesaccording to the present invention will be recognized by those skilledin the art upon reading and understanding the following the DetailedDescription of the Invention as necessary.

Advantageous Effects of Invention

The present invention provides a medicament that can treat or prevent adisease associated with extracellular matrix (ECM) abnormality such asFuchs' endothelial corneal dystrophy, for which only one therapeuticmethod is conventionally keratoplasty, as well as a technique that canrealize eye-drops and the like thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a state that expression of Snail1 and ZEB1 in cornealendothelial cells derived from a Fuchs' endothelial corneal dystrophypatient is promoted. FIG. 1 shows a result of analyzing, by real-timePCR, the expression amount of a gene related to epithelial-mesenchymaltransition (EMT) related to production of an extracellular matrix. TheiFCED was used which was obtained by obtaining and culturing cornealendothelial cells from three patients that led to bullous keratopathy byclinical diagnosis of Fuchs' endothelial corneal dystrophy and underwentDescemet membrane endothelial keratoplasty (DMEK) under the writtenconsent and the approval of Ethics Committee. SV40 and hTERT genes wereintroduced by lentivirus to the cultured corneal endothelial cellsderived from a Fuchs' endothelial corneal dystrophy patient to make animmobilized strain. Corneal endothelial cells cultured from a cornea forresearch imported as a control from a Seattle eye bank were immobilizedby a similar method to make an immobilized cell strain (iHCEC). For theiHCEC and the iFECD, maintenance culture was performed in DMEM+10% FBS.In each graph, the left shows iHCEC, and the right shows iFECD. The Ashows relative expression of Snail1, the B shows relative expression ofSnail2, and the C shows relative expression of ZEB1. By real-time PCR,the significant promotion of expression of Snail1 and ZEB1 wasrecognized in the iFECD in comparison with the iHCEC.

FIG. 2 shows a state that TGF-beta increases expression of Snail1, ZEB1,and in vitro matrix-constituting proteins. FIG. 2 shows a result ofstimulation by TGF-beta, which is known to accelerate expression ofSnail1 and ZEB1, in order to confirm whether the expression promotion ofSnail1 and ZEB1 relates to production of an extracellular matrix. Thewhite shows iHCEC and the black shows iFECD. The A shows the relativeexpression of Snail1, the B shows the relative expression of ZEB1, the Cshows the relative expression of collagen type I, the D shows therelative expression of collagen type IV, the E shows the relativeexpression of collagen type VIII, and the F shows the relativeexpression of Fibronectin. TGF-beta was confirmed to significantlyaccelerate expression of Snail1 and ZEB1 in iFECD (A, B). Then, when thegene expression amount of an extracellular matrix-constituting proteinwas analyzed by real-time PCR, the expression of collagen type I,collagen type IV, collagen type VIII, and Fibronectin was significantlyaccelerated.

FIG. 3 shows a state that TGF-beta accelerates protein production in anin vitro matrix-constituting protein evaluation model. FIG. 3 shows aresult of examining whether an extracellular matrix produced by theiFECD is accelerated by TGF-beta. The iHCEC and the iFECD were culturedin DMEM on Transwell without serum, one week after which they were fixedin a confluent state and HE-stained (The left panel showsphotomicrographs of HE staining, the upper row shows iHCEC, and bottomrow shows iFECD. The left side shows controls and the right side shows aresult of TGF-beta stimulation.). The right graph shows measurementvalues of thickness. In the graph, the left side shows a control, theright side shows a result of stimulation by TGF-beta, the white showsiHCEC, and the black shows iFECD. * and #each show the statisticalsignificance of each other when p<0.05. With regard to the iHCEC andiFECD, it was recognized that the TGF-beta stimulation produced asignificantly thickened extracellular matrix. Further, it was recognizedthat in the presence of TGF-beta, the iFECD produced a significantlythickened extracellular matrix in comparison with the iHCEC. The abovedescription shows that in corneal endothelial cells of a Fuchs'endothelial corneal dystrophy patient, the expression level of Snail1and ZEB1 is high and that the production quantity of an extracellularmatrix in response to the TGF-beta stimulation is significantly higherthan corneal endothelial cells of a healthy subject.

FIG. 4 shows that ZEB1 and Snail1 negatively control the gene expressionof an in vitro matrix-constituting protein. FIG. 4 shows a result ofexamining an effect on the extracellular matrix production bysuppression of Snail1 and ZEB1 using siRNA in order to demonstrate thatthe expression promotion of Snail1 and ZEB1 causes production of anextracellular matrix. The white shows iHCEC and the black shows iFECD.The A to E show a result of Snail1 siRNA and the F to J show a result ofSnail1 siRNA. The A shows a result of ZEB1, the F shows a result ofSNAIL1, the B and G show a result of collagen type I, the C and H show aresult of collagen type IV, the D and I show a result of collagen typeVIII, and the E and J show a result of Fibronectin. * shows statisticalsignificance when p<0.01. It was confirmed that siRNA suppressesexpression of Snail1 and ZEB1 (A, F). Expression suppression of Snail1or ZEB1 by siRNA significantly suppresses expression of collagen type I,collagen type IV, collagen type VIII, and Fibronectin.

FIG. 5 shows a state that ZEB1 or Snail1 negatively control expressionof in vitro matrix-constituting proteins. FIG. 5 shows a result ofexamining expression of collagen type I, collagen type IV, andFibronectin by immunostaining in a similar manner. The left side shows aresult of control siRNA, the middle shows a result of ZEB siRNA, and theright side shows a result of SNAIL1 siRNA. Two upper rows show collagentype I, two middle rows show collagen type IV, two lower rows showFibronectin. Each upper row of the pairs of two rows shows iHCEC andeach lower row thereof shows iFECD. It was confirmed that expressionsuppression of Snail1 or ZEB1 by siRNA also suppresses expression ofcollagen type I, collagen type IV, collagen type VIII, and Fibronectinat a protein level.

FIG. 6 shows that suppression of ZEB1 or Snail1 can suppress in vitromatrix overproduction of Fuchs' endothelial corneal dystrophy cells.FIG. 6 further shows a result of culturing the iHCEC and the iFECD inDMEM on Transwell without serum, and one week later fixing them in aconfluent state and HE-staining (The left panel shows a photomicrographof HE-staining, the upper row shows iHCEC, and the lower row showsiFECD. The left side shows a result of a siRNA control, the middle showsa result of siRNA ZEB1, and the right side shows a result of siRNASNAIL.). The right graph shows measurement values of thickness. In thegraph, the left side shows a siRNA control, the middle shows siRNA ZEB1,the right side shows a result of stimulation by siRNA SNAIL, the whiteshows iHCEC, and the black shows iFECD. * shows the statisticalsignificance when p<0.01. Expression suppression of Snail1 or ZEB1 bysiRNA suppressed extracellular matrix overproduction in the iFECD toresult in the normal level.

FIG. 7 shows a state that TGF-beta signal inhibition suppresses theexpression of Snail1, ZEB1, and an in vitro matrix-constituting protein.FIG. 7 shows a result of inhibiting TGF-beta signal with a TGF-betasignal inhibiting agent SB431542 (0 μM, 1 μM, 3 μM, and 10 μM). Thewhite shows iHCEC and the black shows iFECD. The A shows a result ofSnail1, the B shows a result of ZEB1, the D shows collagen type I, the Eshows collagen type IV, the F shows collagen type VIII, the G showsFibronectin. * shows the statistical significance when p<0.01. As aresult of the TGF-beta signal inhibition, it was recognized by real-timePCR that the expression amount of Snail1 and ZEB1 was significantlydecreased. Further, when the gene expression amount of an extracellularmatrix-constituting protein in the iFCED was analyzed with SB431542 byreal-time PCR, the expression of collagen type I, collagen type IV,collagen type VIII, and Fibronectin was significantly suppressed.

FIG. 8 shows a state that TGF-beta signal inhibition can controlexpression of an in vitro matrix-constituting protein. FIG. 8 shows aresult of examining expression of collagen type I, collagen type IV, andFibronectin by immunostaining, similarly as in FIG. 7 . The left sideshows a result of a control and the right side shows a result ofstimulation by SB431542. Two upper rows show collagen type I, two middlerows show collagen type IV, and two lower rows show Fibronectin. Eachupper row of the pairs of two rows shows iHCEC and each lower rowthereof shows iFECD. It was confirmed that TGF-beta signal inhibitionusing SB431542 also suppresses expression of collagen type I, collagentype IV, collagen type VIII, and Fibronectin in a protein level.

FIG. 9 shows a state that TGF-beta signal inhibition can suppress invitro matrix overproduction of Fuchs' endothelial corneal dystrophycells. FIG. 9 further shows a result of culturing the iHCEC and iFECD inDMEM on Transwell without serum, and one week later fixing them in aconfluent state and HE-staining (In the left panel, the upper row showsiHCEC, and the bottom row shows iFECD. The left side shows a control andthe right side shows a result of stimulation by SB431542.). The rightgraph shows measurement values of thickness. The left side shows acontrol, the right side shows a result of stimulation by SB431542, thewhite shows iHCEC, and the black shows iFECD. * is the statisticalsignificance when p<0.01. TGF-beta signal inhibition using SB431542suppressed extracellular matrix overproduction of iFECD to result in thenormal level.

DESCRIPTION OF EMBODIMENTS

The present invention is described hereinafter. Throughout the entirespecification, a singular expression should be understood asencompassing the concept thereof in the plural form unless specificallynoted otherwise. Thus, singular articles (e.g., “a”, “an”, “the” and thelike in case of English) should also be understood as encompassing theconcept thereof in the plural form unless specifically noted otherwise.Further, the terms used herein should be understood as being used in themeaning that is commonly used in the art, unless specifically notedotherwise. Thus, unless defined otherwise, all terminologies andscientific technical terms that are used herein have the same meaning asthe terms commonly understood by those skilled in the art to which thepresent invention pertains. In case of a contradiction, the presentspecification (including the definitions) takes precedence.

Definition

As used herein, “iFECD” (immobilized or immortalized Fuchs' endothelialcorneal dystrophy) is an abbreviation for an immobilized cell of Fuchs'endothelial corneal dystrophy.

As used herein, “HCEC” (human corneal endothelial cells) is anabbreviation for human corneal endothelial cells. “iHCEC” is anabbreviation for immobilized (or immortalized) human corneal endothelialcells.

As used herein, “transforming growth factor-beta (transforming growthfactor-beta; also referred to as an abbreviated name TGF-beta)” is usedwith the meaning similar to the meaning of those used in the art; andthe transforming growth factor-beta is a homodimer multifunctionalcytokine of a molecular weight of 25 kD, which exhibits various types ofbiological activity. TGF-beta has a role in pathogenesis of a variety ofsclerosing diseases, rheumatoid arthritis, and proliferativevitreoretinopathy, and is greatly involved in hair loss, suppressing theaction of immunocompetent cells, suppressing overproduction of proteaseto prevent lung tissues from being degraded and preventing emphysema,and suppressing the growth of cancer cells, and the like. Three isoformsof TGF-beta exist in humans, namely TGF-beta1 to beta3. TGF-beta isproduced as an inactive latent type with a molecular weight of about 300kD, which is not able to bind to a receptor. TGF-beta is activated on atarget cell surface or in the periphery thereof to become an active typecapable of binding to a receptor, thus exerting the action thereof.

Although it is not desired to be restricted by theories, the action ofTGF-beta in a target cell is regarded as being transmitted by aphosphorylation pathway of a set of proteins for performing informationtransmission, referred to as Smad. First, when active TGF-beta is boundto a type II TGF-beta receptor present on a surface of a target cell, areceptor complex is formed which consists of two molecules of a type IIreceptor and two molecules of a type I TGF-beta receptor, and the typeII receptor phosphorylates the type I receptor. Next, the phosphorylatedtype I receptor phosphorylates Smad2 or Smad3, and the phosphorylatedSmad2 or Smad3 forms a complex with Smad4, and the complex transfers toa nucleus, binds to a target sequence referred to as CAGA box, which ispresent in a target gene promoter region, and induces transcriptionalexpression of a target gene together with a coactivator.

The transforming (transformation) growth factor-beta (TGF-beta) signaltransduction pathway is capable of regulating many cell activities, suchas cell growth and differentiation, growth arrest, apoptosis, andepithelial-to-mesenchymal conversion (EMT), by regulation of a targetgene thereof. TGF-beta family members, including the TGF-beta itself(such as TGF-beta 1, TGF-beta 2 and TGF-beta 3), activin and bonemorphogenic protein (BMP), are strong regulating agents for cell growth,differentiation, migration and apoptosis.

The TGF-beta is a protein of about 24 kD, which is produced by manycells including B lymphocyte, T lymphocyte and activated macrophage, andby many other cell types. Effects of TGF-beta to immune systems includeIL-2 receptor induction, inhibition of IL-1 induced thymic cell growth,and blocking of IFN-gamma-induced macrophage activation. The TGF-beta isthought to be involved in a variety of pathological conditions (Borderet al., (1992) J. Clin. Invest. 90:1), and is sufficiently supported tofunction as either a tumor inhibitory substance or a tumor promoter.

TGF-beta mediates the signaling thereof by two serine/threonine kinasecell surface receptors, TGF-betaRII and ALK5. TGF-beta signaling isinitiated by ligand-induced receptor dimerization, which allowsTGF-betaRII to phosphorylate an ALK5 receptor. The phosphorylationthereof is such that ALK5 kinase activity is activated and the activatedALK5 then phosphorylates a downstream effector Smad protein (vertebratehomologue of MAD or “Mothers against DPP (decapentaplegic)” protein),Smad2 or 3. The p-Smad2/3 complex with Smad4 enters a nucleus toactivate the transcription of a target gene.

Smad3 is a member of an R-Smad (receptor-activated Smad) subgroup ofSmad, and is a direct mediator of activation of transcription by aTGF-beta receptor. TGF-beta stimulation causes phosphorylation andactivation of Smad2 and Smad3, which forms a complex with Smad4 (“commonSmad” or “co-Smad” in vertebrates), which is accumulated together with anucleus to regulate the transcription of a target gene. R-Smad islocalized at a cytoplasm, and forms a complex with a co-Smad throughligand-induced phosphorylation by a TGF-beta receptor; and the complexmoves to a nucleus, which then regulates gene expression that isassociated with chromatin and a cooperative transcription factor. Smad6and Smad7 are each inhibitory Smad (“I-Smad”), that is, they aretranscriptionally induced by TGF-beta and function as an inhibitingagent for TGF-beta signaling (Feng et al., (2005) Annu. Rev. Cell. Dev.Biol. 21:659). Smad6/7 inhibits the receptor-mediated activation ofR-Smad to exert their inhibitory effect; and they are associated with atype I receptor, which competitively prevents mobilization andphosphorylation of R-Smad. Smad6 and Smad7 are known to replenish E3ubiquitin ligase, which causes ubiquitination and degradation of Smad6/7interactive protein.

With regard to the TGF-beta signal transduction pathway, another pathwayadditionally exists which is transmitted by BMP-7 or the like, which isregarded as exhibiting functions via ALK-1/2/3/6 and then via Smad1/5/8.With regard to the TGF-beta signal transduction pathway, also see J.Massagu'e, Annu. Rev. Biochem. 1998. 67: 753-91; Vilar J M G, Jansen R,Sander C (2006) PLoS Comput Biol 2(1):e3; Leask, A., Abraham, D. J.FASEB J. 18, 816-827 (2004); Coert Margadant & Arnoud Sonnenberg EMBOreports (2010) 11, 97-105; Joel Rosenbloom et al., Ann Intern Med. 2010;152: 159-166 and the like.

As used herein, “transforming growth factor (TGF)-beta signal inhibitingagent” refers to any factor that inhibits TGF signaling. When TGF-betais counteracted, the agent responsible may be referred to as anantagonist. However, in the case of the present invention, the TGF-betaantagonist is encompassed by the TGF-beta signal inhibiting agent. Sincethis inhibiting agent is commonly a substance, a “TGF-beta signalinhibitory substance” may be interchangeably used with a “TGF-betasignal inhibiting agent”.

Therefore, the TGF-beta signal inhibiting agent used in the presentinvention typically includes, without limitation, an antagonist ofTGF-beta, an antagonist of a receptor of TGF-beta, and an inhibitingagent of Smad3, a ligand trap (an antibody against a ligand, a decoyreceptor), an antisense oligonucleotide, a TGF-beta receptor kinaseinhibiting agent, a peptide aptamer, siRNA, shRNA, and the like (referto Connolly E., et al., Int. J. Biol. Sci. 2012; 8(7): 964-978, FIG. 3 ,and the like).

Exemplary TGF-beta signal inhibiting agent that may be used in thepresent invention can include, without limitation, SB431542(4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)]-1H-imidazol-2-yl]benzamide),BMP-7, anti-TGF-beta antibody, anti-TGF-beta receptor antibody, siRNA ofTGF-beta, siRNA of TGF-beta receptor, antisense oligonucleotide ofTGF-beta,6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone,A83-01(3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide),Stemolecule™ TLK inhibitor(2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine),Stemolecule™ BMP inhibitor LDN-193189(6-(4-(piperidine-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine),SD-208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine),LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline),pharmaceutically acceptable salts and a solvates thereof, and solvatesof the pharmaceutically acceptable salts, and the like.

Other TGF-beta signal inhibiting agents include, without limitation, amonoclonal antibody and a polyclonal antibody to one or more isoforms ofTGF-beta (U.S. Pat. No. 5,571,714; also see International PublicationNo. WO 97/13844 and International Publication No. WO 00/66631), TGF-betareceptor, a soluble form of such a receptor (e.g., soluble TGF-beta typeIII receptor), or an antibody directed to a TGF-beta receptor (U.S. Pat.Nos. 5,693,607, 6,001,969, 6,010,872, 6,086,867, 6,201,108;International Publication No. WO 98/48024; International Publication No.WO 95/10610; International Publication No. WO 93/09228; InternationalPublication No. WO 92/00330), latent and associated peptide(International Publication No. WO 91/08291), large latent TGF-beta(International Publication No. WO 94/09812), fetuin (U.S. Pat. No.5,821,227), other proteoglycan such as decorin and biglycan,fibromodulin, lumican, and endoglin and the like (InternationalPublication No. WO 91/10727; U.S. Pat. Nos. 5,654,270, 5,705,609,5,726,149; 5,824,655; International Publication No. WO 91/04748; U.S.Pat. Nos. 5,830,847, 6,015,693; International Publication No. WO91/10727; International Publication No. WO 93/09800; and InternationalPublication No. WO 94/10187), somatostatin (International PublicationNo. WO 98/08529), mannose-6-phosphoric acid or mannose-1-phosphoric acid(U.S. Pat. No. 5,520,926), prolactin (International Publication No. WO97/40848), insulin-like growth factor II (International Publication No.WO 98/17304), IP-10 (International Publication No. WO 97/00691),Arg-Gly-Asp-containing peptide (Pfeffer, U.S. Pat. No. 5,958,411;International Publication No. WO 93/10808), plants, fungi and bacteriaextracts (EP-A-813875; Japanese Laid-Open Publication No. 8-119984; andMatsunaga et al., U.S. Pat. No. 5,693,610), antisense oligonucleotide(U.S. Pat. Nos. 5,683,988; 5,772,995; 5,821,234, 5,869,462; andInternational Publication No. WO 94/25588), protein associated withTGF-beta signaling including Smad and MAD (EP-A-874046; InternationalPublication No. WO 97/31020; International Publication No. WO 97/38729;International Publication No. WO 98/03663; International Publication No.WO 98/07735; International Publication No. WO 98/07849; InternationalPublication No. WO 98/45467; International Publication No. WO 98/53068;International Publication No. WO 98/55512; International Publication No.WO 98/56913; International Publication No. WO 98/53830; InternationalPublication No. WO 99/50296; U.S. Pat. Nos. 5,834,248; 5,807,708; and5,948,639), Ski and Sno (Vogel, 1999, Science, 286:665; and Stroscheinet al., 1999, Science, 286:771 to 774), one or more single-strandedoligonucleotide aptamers or an expression plasmid encoding them,suitable for inhibiting or interfering the binding of TGF-beta to areceptor of the same origin, and any mutant, fragment or derivative of amolecule identified above, which retains an ability to inhibit theactivity of TGF-beta. The TGF-beta inhibiting agent may be a TGF-betaantagonist, and may be a human monoclonal antibody or a humanizedmonoclonal antibody (or F(ab)₂ fragment, Fv fragment, single chainantibody, and other forms or fragments of an antibody retaining theability to bind to TGF-beta, a fragment thereof or the like), whichblocks TGF-beta binding to the receptor. The TGF-beta receptor and aTGF-beta binding fragment, and in particular a soluble fragment, of aTGF-beta receptor are TGF-beta antagonists which are useful in themethod according to the present invention. In a certain embodiment, aninhibiting agent preferable for TGF-beta functions is a soluble TGF-betareceptor, and in particular, a TGF-beta type II receptor (TGFBIIR) or aTGF-beta type III receptor (TGFBIIIR or betaglycan) including, forexample, an extracellular domain of TGFBIIR or TGFBIIIR, preferably arecombinant soluble TGF-beta receptor (rsTGFBIIR or rsTGFBIIIR). TheTGF-beta receptor and a TGF-beta binding fragment of the TGF-betareceptor, in particular a soluble fragment, are TGF-beta antagonistsuseful in the method according to the present invention. TGF-betareceptors and nucleic acids encoding them are sufficiently known in theart. A nucleic acid sequence encoding TGF-beta type 1 receptor isdisclosed in GenBank accession number L15436 and U.S. Pat. No. 5,538,892(Donahoe et al.). A nucleic acid sequence of a TGF-beta type 2 receptoris publicly available under GenBank accession number AW236001, AI35790,AI279872, AI074706, and AA808255. A nucleic acid sequence of a TGF-betatype 3 receptor is also publicly available under GenBank accessionnumber NM003243, AI887852, AI817295, and AI681599.

In addition, still other TGF-beta signal inhibiting agents orantagonists and methods for producing them, are sufficiently known inthe art, in addition to many of those that are currently underdevelopment. Any of effective TGF-beta antagonists may be useful in themethod according to the present invention, and thus, specific TGF-betasignal inhibiting agents or antagonists used are not those with limitedcharacteristics. Examples of such antagonists include monoclonal andpolyclonal antibodies to TGF-beta of one or more isotypes (U.S. Pat. No.5,571,714 and International Publication No. WO 97/13844), TGF-betareceptor, a fragment thereof, a derivative thereof, and an antibody to aTGF-beta receptor (U.S. Pat. Nos. 5,693,607, 6,008,011, 6,001,969 and6,010,872, and International Publication No. WO 92/00330, InternationalPublication No. WO 93/09228, International Publication No. WO 95/10610,and International Publication No. WO 98/48024); latency-associatedpeptide (latency associated peptide; International Publication No. WO91/08291), large lacent TGF-beta (International Publication No. WO94/09812), fetuin (U.S. Pat. No. 5,821,227), other proteoglycan such asdecorin and biglycan, fibromodulin, lumican, endoglin, and the like(U.S. Pat. Nos. 5,583,103, 5,654,270, 5,705,609, 5,726,149, 5,824,655,5,830,847, 6,015,693, and International Publication No. WO 91/04748,International Publication No. WO 91/10727, International Publication No.WO 93/09800 and International Publication No. WO 94/10187).

Further examples of such an antagonist include a host of other proteinsassociated with TGF-beta signaling, including somatostatin(International Publication No. WO 98/08529), mannose-6-phosphoric acidor mannose-1-phosphoric acid (U.S. Pat. No. 5,520,926), prolactin(International Publication No. WO 97/40848), insulin-like growth factorII (International Publication No. WO 98/17304), IP-10 (InternationalPublication No. WO 97/00691), arginine (arg)-glycine (gly)-aspartic acid(asp)-containing peptide (U.S. Pat. No. 5,958,411 and InternationalPublication No. WO 93/10808), plants, fungi and bacteria extracts(European Patent Application Publication No. 813875, Japanese Laid-OpenPublication No. 8-119984 and U.S. Pat. No. 5,693,610), antisenseoligonucleotide (U.S. Pat. Nos. 5,683,988, 5,772,995, 5,821,234 and5,869,462, and International Publication No. WO 94/25588), and Smad andMAD (European Patent Application No. EP874046, International PublicationNo. WO 97/31020, International Publication No. WO 97/38729,International Publication No. WO 98/03663, International Publication No.WO 98/07735, International Publication No. WO 98/07849, InternationalPublication No. WO 98/45467, International Publication No. WO 98/53068,International Publication No. WO 98/55512, International Publication No.WO 98/56913, International Publication No. WO 98/53830 and InternationalPublication No. WO 99/50296, and U.S. Pat. Nos. 5,834,248, 5,807,708 and5,948,639), and Ski and Sno (G. Vogel, Science, 286:665(1999) andStroschein et al., Science, 286:771-74(1999)), and any fragment andderivative of the above-mentioned molecule retaining the ability toinhibit the activity of TGF-beta.

The TGF-beta antagonists suitable for the use in the present inventionalso include a functional mutant, a mutant, a derivative, and ananalogue of the aforementioned TGF-beta antagonist so long as theirability of inhibiting the amount or activity of TGF-beta is retained.The “mutant”, “derivative”, and “analogue” as used herein refers to amolecule having a form or structure similar to that of their parentcompound, and retaining an ability to work as a TGF-beta antagonist. Forexample, any of the TGF-beta antagonists disclosed in the presentspecification may be crystallized, and useful analogues may bereasonably designed based on sites that have a role in forming (one ormore) active sites. Instead, those skilled in the art can alter afunctional group of known antagonists, or can screen such an alteredmolecule with regard to an increase of activity, half-life,bioavailability, or other desirable characteristics, without unnecessaryexperiments. When the TGF-beta antagonist is a polypeptide, a fragmentand variant of the polypeptide may be produced to increase the ease ofdelivery, activity, half-life and the like (e.g., humanized antibodiesor functional antibody fragments discussed above). In consideration ofthe technical level in the art for producing synthetic and recombinantpolypeptides, such a variant may be achieved without unnecessaryexperiments. Those skilled in the art may also design a novel inhibitingagent based on knowledge on a crystal structure and/or active site ofthe TGF-beta inhibiting agent as described herein. A polypeptideinhibiting agent, such as a soluble TGF-beta receptor, may beeffectively introduced through gene transfer. Accordingly, a certainembodiment for the method according to the present invention includesuse of a vector suitable for expression of a TGF-beta receptor or abinding partner, preferably a soluble receptor or a soluble bindingpartner. In a preferable embodiment, administration of a solubleTGF-beta antagonist can be achieved by gene transfer which uses a vectorcomprising a cDNA encoding a soluble antagonist or a cDNA encoding anextracellular domain of a TGF-beta type II receptor (rsTGFBIIR) or aTGF-beta type III receptor (rsTGFBIIIR). This vector causes an in situexpression of a soluble TGF-beta antagonist in a cell which istransfected using the vector, inhibits the activity of TGF-beta, andsuppresses TGF-beta-mediated fibrogenesis. Any suitable vector can beused. Preferable vectors include an adenovirus vector, a lentivirusvector, an Epstein-Barr virus (EBV) vector, an adeno-associated virus(AAV) vector, and a retrovirus vector, developed for the purpose of genetransfer. Other non-vector methods for gene transfer may also be used,such as lipid/DNA complex, protein/DNA conjugate and naked DNA transfermethods. Further suitable TGF-beta antagonists developed for deliveryvia adenovirus gene transfer include, without limitation, a chimericcDNA encoding an extracellular domain of a TGF-beta type II receptor,fused to an Ig Fc domain (Isaka et al., 1999, Kidney Int., 55:pp. 465 to475), an adenovirus gene transfer vector of a dominant negative mutantof a TGF-beta type II receptor (Zhao et al., 1998, Mech. Dev., 72:pp. 89to 100), and an adenovirus gene transfer vector of decorin, which is aTGF-beta binding proteoglycan (Zhao et al., 1999, Am. J. Physiol., 277:pp. L412 to L422). Adenovirus-mediated gene transfer has extremely highefficiency compared to other gene delivery manners.

The TGF-beta receptor and a TGF-beta binding fragment, a solublefragment and the like of the TGF-beta receptor are TGF-beta antagonistsuseful in the present invention. The TGF-beta receptors and nucleicacids encoding them are sufficiently known in the art. The nucleic acidsequence encoding the TGF-beta type 1 receptor is disclosed in GenBank,accession number L15436 and U.S. Pat. No. 5,538,892 by Donahoe et al. Anucleic acid sequence of the TGF-beta type 2 receptor is also publiclyavailable under GenBank accession number AW236001; AI35790; AI279872;AI074706; and AA808255. A nucleic acid sequence of the TGF-beta type 3receptor is also publicly available under GenBank accession numberNM003243; AI887852; AI817295; and AI681599. In one exemplary embodiment,the TGF-beta antagonist is an antibody which blocks TGF-beta binding toa receptor thereof, or to fragments thereof such as a F(ab)₂ fragment, aFv fragment, a single-stranded antibody, and other “antibody” typesretaining the ability to bind to TGF-beta. The antibody thereof may bechimerized or humanized. Herein, the chimerized antibody includes aconstant region of a human antibody, and a variable region of non-humanantibodies such as a murine antibody. The humanized antibody includes aconstant region and a framework variable region (i.e., variable regionsother than hypervariable regions) of a human antibody, and ahypervariable region of non-human antibodies such as a murine antibody.As a matter of course, the antibody may be any other types of antibodyderivatives, such as human antibodies selected from a phage displaysystem or produced from a XenoMouse.

Findings related to Smad are increasing. TGF-beta signal transductionpathway is initiated when this molecule binds to a heterodimer cellsurface complex consisting of a serine/threonine kinase receptor of typeI (TbRI) and type II (TbRII) and induces this heterodimer cell surfacecomplex. Then, the heterodimer receptor transmits said signal throughphosphorylation of a target Smad protein in the downstream. As describedabove, there are three functional classes for the Smad protein, and theyare, for example, Smad (R-Smad) restricted by a receptor such as Smad2and Smad3, a co-mediator (Co-Smad) which is also referred to as Smad4,and an inhibitory Smad (I-Smad). Followed by the phosphorylation by theheterodimer receptor complex, this R-Smad forms a complex with thisCo-Smad, moves to said nucleus, and working together with otherrespective proteins, they regulate transcription of the target gene(Derynck, R., et al. (1998) Cell 95: 737-740); Massague, J. and Wotton,D. (2000) EMBO J. 19:1745). A nucleotide sequence and an amino acidsequence of human Smad3 are disclosed in, for example, GenBank AccessionNo. gi:42476202. A nucleotide sequence and an amino acid sequence ofmurine Smad3 is disclosed in, for example, GenBank Accession No. gi:31543221. As described above, TGF-beta stimulation providesphosphorylation and activation of Smad2 and Smad3, which form a complexwith Smad4 (also referred to as “common Smad” or “co-Smad”), and thecomplex is accumulated with a nucleus to regulate the transcription ofthe target gene. Accordingly, the TGF-beta signal inhibition may also beachieved by inhibition of Smad2, 3 or co-Smad (Smad4). The R-Smad islocalized in a cytoplasm, and forms a complex with a co-Smad throughligand-induced phosphorylation by a TGF-beta receptor to move to anucleus, in which they regulate gene expression associated with achromatin and a cooperative transcription factor. Thus, TGF-beta signalinhibition can also be achieved by inhibiting R-Smad either directly orindirectly. Smad6 and Smad7 are inhibitory Smad (I-Smad), and that is,they are transcriptionally induced by TGF-beta to function as aninhibiting agent of TGF-beta signaling (Feng et al., (2005) Annu. Rev.Cell. Dev. Biol. 21: 659). Smad6/7 prevents receptor-mediated activationof R-Smad, thereby exerting their inhibitory effect. They are associatedwith a type I receptor, which competitively inhibits mobilization andphosphorylation of R-Smad. Smad6 and Smad7 are known to replenish E3ubiquitin ligase, which causes ubiquitination and degradation of Smad6/7interactive protein. Thus, Smad6 and 7 can function as a TGF-beta signalinhibiting agent in the present invention.

The inhibiting agents of Smad3 that may be used in the present inventioncan include, without limitation, antisense nucleotide, siRNA, antibodyand the like, and in addition,6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone,and the like commercially available from Calbiochem, as a low-molecularcompound.

As used herein, “substance (e.g., nucleic acid) for suppressingexpression (of TGF-beta or the like)” is not particularly limited solong as such a substance is a substance which suppresses transcriptionof mRNA of a target gene, a substance which degrades a transcribed mRNA(e.g., nucleic acid), or a substance (e.g., nucleic acid) whichsuppresses translation of protein from mRNA. As to the substances,exemplified are siRNA, antisense oligonucleotide, and ribozyme as wellas nucleic acids of expression vectors. Among them, siRNA and anexpression vector thereof are preferable, and siRNA is particularlypreferable. “Substance which suppresses expression of a gene” includes,in addition to those described above, protein, peptide, and other smallmolecules. Note that a target gene in the present invention means anygene that is associated with a TGF-beta signal transduction pathway.

As to a method for inhibiting the expression of a specific endogenousgene, such as TGF-beta, that is targeted in the present invention, amethod utilizing an antisense technique is well known to those skilledin the art. As to actions for an antisense nucleic acid to inhibit theexpression of a target gene, there are a plurality of factors asfollows. Specifically, such factors are: inhibition of transcriptinitiation due to triplex formation; inhibition of transcription due tohybrid formation with a site where an open loop structure is locallyformed due to RNA polymerase; inhibition of transcription due to hybridformation with an RNA whose synthesis is in progress; splicinginhibition due to hybrid formation at a junction of intron and exon;splicing inhibition due to hybrid formation with spliceosome formingsite; transfer inhibition from a nucleus to cytoplasm due to hybridformation with mRNA; splicing inhibition due to hybrid formation with acapping site or a poly (A) addition site; inhibition of translationinitiation due to hybrid formation with a translation initiation factorbinding site; translational inhibition due to hybrid formation with aribosome binding site near an initiation codon; elongation inhibition ofa peptide chain due to hybrid formation with a polysome binding site ora translation region of mRNA; and gene expression inhibition due tohybrid formation with a interaction site of a nucleic acid and aprotein, and the like. As such, an antisense nucleic acid inhibits avariety of processes, such as transcription, splicing or translation, toinhibit the expression of a target gene (Hirashima and Inoue, ShinseiKagaku Jikken Kouza [New Chemical Experiment Course] 2, Nucleic Acid, IVIdenshi no Fukusei to Hatsugen [Duplication and Expression of Gene],Edited by the Japanese Biochemical Society, Tokyo Kagaku Dozin, 1993,319-347).

The antisense nucleic acid used in the present invention may inhibit theexpression and/or function of a gene (nucleic acid) encoding a member orthe like of a signal transduction pathway of the above-mentionedTGF-beta by any of the above-mentioned actions. In one embodiment, it isconsidered to be effective for the translation inhibition of a gene whenan antisense sequence complementary to a non-translation region near 5′terminal of mRNA of a gene encoding the above-mentioned TGF-beta or thelike is designed. In addition, it is possible to use a sequencecomplementary to a coding region or a 3′ non-translation region. Assuch, the translation region of a gene encoding the above-mentionedTGF-beta or the like as well as a nucleic acid including an antisensesequence of a sequence of a non-translation region are included in theantisense nucleic acid that are used in the present invention. Theantisense nucleic acid used is connected to a downstream of anappropriate promoter, and is preferably connected to a sequenceincluding a transcription termination signal on the side closer to 3′. Anucleic acid prepared in such a manner can be transformed into a desiredanimal (cell) using a publicly known method. While the sequence of theantisense nucleic acid is preferably a sequence complementary to a gene,or a part thereof, encoding TGF-beta or the like of an animal (cell) tobe transformed, it does not have to be completely complementary so longas the sequence can effectively suppress the expression of genes. Thetranscribed RNA preferably has 90% or more, and most preferably 95% ormore, complementarity to a transcription product of a target gene. Inorder to effectively inhibit the expression of a target gene using anantisense nucleic acid, the length of the antisense nucleic acid ispreferably at least 12 bases or more but less than 25 bases long.However, the antisense nucleic acid according to the present inventionis not necessarily limited to this length, and the antisense nucleicacid may be, for example, 11 bases or less, 100 bases or more, or 500bases or more. While the antisense nucleic acid may be composed of DNAonly, it may also include nucleic acids other than DNA, such as lockednucleic acid (LNA). In one embodiment, the antisense nucleic acid usedin the present invention may be a LNA-containing antisense nucleic acidincluding LNA at the 5′ terminal, and LNA at the 3′ terminal.Furthermore, in an embodiment where an antisense nucleic acid is used inthe present invention, an antisense sequence can be designed based on anucleic acid sequence, such as TGF-beta, using a method described inHirashima and Inoue, Shinsei Kagaku Jikken Kouza [New ChemicalExperiment Course] 2, Nucleic Acid, IV Idenshi no Fukusei to Hatsugen[Duplication and Expression of Gene], Edited by the Japanese BiochemicalSociety, Tokyo Kagaku Dozin, 1993, 319-347, for example.

The inhibition of expression of TGF-beta or the like can also beperformed by using ribozyme, or DNA encoding ribozyme. The ribozymerefers to a RNA molecule having catalytic activity. There are varioustypes of ribozymes having various types of activities, and researchesfocusing on especially a ribozyme as an enzyme for cleaving RNA has madeit possible to design a ribozyme for cleaving RNA in a site-specificmanner. While ribozymes include those with 400 nucleotides or more insize, such as group I intron type and M1 RNA included in RNase P, thereare also such ribozymes having an activity domain of as many as 40nucleotides, such as those referred to as hammer head type and hairpintype (Makoto Koizumi and Eiko Ohtsuka, Tanpakushitu Kakusan Kouso[Protein Nucleic Acid Enzyme], 1990, 35, 2191).

For example, the self-cleavage domain of the hammer head type ribozymecleaves the side closer to 3′ of C15 in a sequence referred to asG13U14C15, and the base-pair formation of U14 and A9 is considered to beimportant for the activity thereof; and it is indicated that cleavagecan be made by A15 or U15, instead of C15 (Koizumi, M. et al., FEBSLett, 1988, 228, 228). If a ribozyme is designed in which a substancebinding site is complementary to a RNA sequence near a target site, arestriction-enzymic RNA cleavage ribozyme can be created whichrecognizes a sequence such as UC, UU or UA in a target RNA (Koizumi, M.et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Ohtsuka,Tanpakushitu Kakusan Kouso [Protein Nucleic Acid Enzyme], 1990, 35,2191., Koizumi, M. et al., Nucl. Acids Res., 1989, 17, 7059).

In addition, hairpin type ribozyme are also useful for the purpose ofthe present invention. Such a ribozyme is found in, for example, anegative strand of a satellite RNA of tobacco ringspot virus (Buzayan, JM., Nature, 1986, 323, 349.). It is indicated that a target-specific RNAcleavage ribozyme can be created from hairpin type ribozyme (Kikuchi, Y.& Sasaki, N., Nucl. Acids Res, 1991, 19, 6751., Kikuchi, Yo, Kagaku toSeibutu [Chemistry and Living Organism], 1992, 30, 112.). As such, atranscription product of a gene encoding TGF-beta or the like isspecifically cleaved using ribozyme, so that the expression of the genecan be inhibited.

Suppression of expression of an endogenous gene of TGF-beta or the likecan also be performed by RNA interference (hereinafter, abbreviated as“RNAi”) using a double-stranded RNA having a sequence identical orsimilar to a target gene sequence. With regard to the RNAi, whendouble-stranded RNA (dsRNA) is taken directly into a cell, expression ofa gene having a sequence homologous to the dsRNA is suppressed, which isa method that is currently attracting attention. In mammalian cells, ashort strand dsRNA (siRNA) is used so that RNAi can be induced. Incomparison with knockout mice, RNAi has many advantages, such as highstability of an effect, easy experimentation, and inexpensive cost. ThesiRNA will be described in detail in a different part of the presentspecification.

As used herein, “siRNA” refers to an RNA molecule having adouble-stranded RNA moiety consisting of 15 to 40 bases, and the siRNAhas a function of cleaving mRNA of a target gene having a sequencecomplementary to an antisense strand of said siRNA and suppressing theexpression of the target gene. More specifically, the siRNA according tothe present invention is an RNA including a double-stranded RNA moietyconsisting of a sense RNA chain consisting of a sequence homologous to acontiguous RNA sequence in mRNA of TGF-beta or the like, and anantisense RNA chain consisting of a sequence complementary to the senseRNA sequence. The manufacturing and designing of the siRNA and a mutantsiRNA to be described below are within the scope of the ability of thoseskilled in the art. The concept of selecting any contiguous RNA regionof mRNA, which is a transcription product of a sequence of TGF-beta orthe like, and creating a double-stranded RNA corresponding to the regionis merely a matter that those skilled in the art can perform within thenormal creative ability of them. Furthermore, the concept of selecting asiRNA sequence with a more powerful RNAi effect from an mRNA sequence,which is a transcription product of the subject sequence, can beappropriately performed by those skilled in the art using a publiclyknown method. Furthermore, if one of the strands is identified, it iseasy for those skilled in the art to determine a base sequence of theother strand (complementary strand). Those skilled in the art canappropriately create siRNA using a commercially available nucleic acidsynthesizing machine. In addition, synthesis entrustment service can begenerally used for desired RNA synthesis.

The length of the double-stranded RNA moiety is, as a base, 15 to 40bases, preferably 15 to 30 bases, more preferably 15 to 25 bases, stillmore preferably 18 to 23 bases, and most preferably 19 to 21 bases. Itis understood that the upper and lower limits thereof are not limited tothe specified ones, but the limits can be any combinations of the listedones. As to a terminal structure of a sense strand or antisense strandof siRNA, there is no particular limitation, and it can be appropriatelyselected depending on the purpose. For example, the terminal structuremay be the one having a flush terminal or the one having protrudingterminal (overhang), and the type with protruded 3′ terminal ispreferable. A siRNA having an overhang consisting of several bases,preferably 1 to 3 bases, and still preferably 2 bases, at the 3′terminal of the sense RNA strand and antisense RNA strand often has agreat effect of inhibiting the expression of a target gene, which ispreferable. The type of the bases of overhang is not particularlyrestricted, and the type can be either a base constituting an RNA or abase constituting a DNA. Preferable overhang sequences can include dTdT(2 bp deoxy T) at the 3′ terminal, and the like. For example, preferablesiRNAs include, without limitation, those in which dTdT (2 bp deoxy T)is added to 3′ terminal of the sense and antisense strands of all thesiRNA.

Furthermore, it is also possible to use a siRNA in which one to severalnucleotides are deleted, substituted, inserted and/or added in either orboth of the sense strand and antisense strand of the above-mentionedsiRNA. In this regard, the concept of one to several bases is notparticularly limited, but it is preferably 1 to 4 bases, stillpreferably 1 to 3 bases, most preferably 1 to 2 bases. Specific examplesof the subject mutation include, without limitation, those in which thenumber of bases at the 3′ overhang moiety is from 0 to 3, those in whichthe base sequence of the 3′-overhang moiety is changed to another basesequence, those in which the length of the above-mentioned sense RNAstrand and antisense RNA strand is different by 1 to 3 bases due to theinsertion, addition or deletion of bases, those in which the base in asense strand and/or antisense strand is substituted with another base,and the like. However, it is necessary for the sense strand and theantisense strand to be able to hybridize in these mutant siRNAs, and itis necessary for these mutant siRNAs to have an ability to inhibit geneexpression equivalent to siRNAs that do not have mutation.

Furthermore, the siRNA may be a siRNA (Short Hairpin RNA; shRNA) inwhich one of the terminals has a molecule of a closed structure, such asa hairpin structure. The shRNA is a sense strand RNA of a specificsequence of a target gene, an antisense strand RNA consisting of asequence complementary to the sense strand sequence, and a RNA includinga linker sequence for connecting the both strands thereof, wherein thesense strand moiety and the antisense strand moiety hybridize to form adouble-stranded RNA moiety.

The siRNA desirably does not exhibit a so-called off-target effect whenclinically used. The off-target effect refers to an effect forsuppressing the expression of another gene with partially homology tothe siRNA used, other than the target gene. In order to avoid theoff-target effect, it is possible to confirm that a candidate siRNA doesnot have cross reactivity using DNA microarray or the like in advance.Furthermore, it is possible to avoid the off-target effect by confirmingas to whether there is a gene including a moiety having high homologywith a sequence of a candidate siRNA, other than a target gene, usingpublicly known database provided by NCBI (National Center forBiotechnology Information) or the like.

In order to create the siRNA according to the present invention, apublicly known method, such as a method by chemical synthesis and amethod using a gene recombination technique, can be appropriately used.With a method by synthesis, a double-stranded RNA can be synthesizedbased on sequence information, using an ordinary method. In addition, ina method using a gene recombination technique, it is also possible tocreate such a siRNA by constructing an expression vector encoding asense strand sequence and an antisense strand sequence and introducingthe vector into a host cell, and then obtaining a sense strand RNA andan antisense strand RNA, each of which is produced by transcription.Furthermore, it is possible to create a desired double-stranded RNA byexpressing a shRNA, which includes a sense strand of a specific sequenceof a target gene, an antisense strand consisting of a sequencecomplementary to the sense strand sequence, and a linker sequence forconnecting the both strands, and which forms a hairpin structure.

With regard to the siRNA, all or part of the nucleic acids constitutingthe siRNA may be a natural nucleic acid or a modified nucleic acid solong as such a nucleic acid has an activity to suppress the expressionof a target gene.

The siRNA according to the present invention does not necessarily haveto be a pair of double-stranded RNAs to a target sequence, and it may bea mixture of a plurality (the “plurality” is not particularly limited,but preferably refers to a small number of about 2 to 5) ofdouble-stranded RNAs to a region which includes a target sequence. Inthis regard, those skilled in the art can appropriately create siRNA, asa nucleic acid mixture corresponding to a target sequence, using acommercially available nucleic acid synthesizing machine and DICERenzyme; and as to synthesis of a desired RNA, synthesis entrustmentservice can be generally used. Note that the siRNA according to thepresent invention includes a so-called “cocktail siRNA”. Furthermore,note that the siRNA according to the present invention is such that notall the nucleotides have to be a ribonucleotide (RNA). Specifically, inthe present invention, one or plurality of ribonucleotides constitutinga siRNA may be a corresponding deoxyribonucleotide. The term“corresponding” refers to being the same base type (adenine, guanine,cytosine, thymine (uracil)) although the structure of the sugar portionis different. For example, a deoxyribonucleotide corresponding to aribonucleotide having adenine refers to a deoxyribonucleotide havingadenine.

Furthermore, a DNA (vector) which may express the above-mentioned RNAaccording to the present invention is also included in a preferredembodiment of a nucleic acid which may suppress expression of TGF-betaor the like. For example, the DNA (vector) which may express theabove-mentioned double-stranded RNA according to the present inventionis such a DNA having a structure in which DNA encoding one of thestrands of the double-stranded RNA and a DNA encoding the other of thestrands of the double-stranded RNA are connected to a promoter so thateach of the DNAs is capable of being expressed. The above-mentioned DNAaccording to the present invention can be appropriately created by thoseskilled in the art using a general genetic engineering technique. Morespecifically, the expression vector according to the present inventioncan be created by appropriately inserting the DNA encoding RNA accordingto the present invention, into a variety of publicly known expressionvectors.

In the present invention, a modified nucleic acid may be used for thenucleic acid for suppressing the expression of a target gene. Themodified nucleic acid means a nucleic acid in which modification isprovided at a nucleoside (base moiety, sugar moiety) and/or aninter-nucleoside binding site, and that has a structure different fromthat of a natural nucleic acid. “Modified nucleoside”, which constitutesa modified nucleic acid, includes, for example, abasic nucleoside;arabinonucleoside, 2′-deoxyuridine, alpha-deoxyribonucleoside,beta-L-deoxyribonucleoside, nucleoside having other sugar modification;peptide nucleic acid (PNA), phosphate group-binding peptide nucleic acid(PHONA), locked nucleic acid (LNA), morpholino nucleic acid and thelike. The above-mentioned nucleoside having sugar modification includesnucleosides having a substituted pentose, such as 2′-O-methylribose,2′-deoxy-2′-fluororibose, 3′-O-methylribose, and the like;1′,2′-deoxyribose; arabinose; a substituted arabinose sugar; and ahexose, and sugar modification of an alpha-anomer. These nucleosides maybe a modified base in which the base moiety is modified. Such modifiedbases include, for example, pyrimidine, such as 5-hydroxycytosine,5-fluorouracil, 4-thiouracil, and the like; purine, such as6-methyladenine, 6-thioguanosine, and the like; and other heterocyclicbases.

“Modified inter-nucleoside binding”, which constitutes a modifiednucleic acid, includes non-natural inter-nucleoside binding, such asalkyl linker, glyceryl linker, amino linker, poly(ethylene glycol)binding, inter-methyl phosphonate nucleoside binding;methylphosphonothioate, phosphotriester, phosphothiotriester,phosphorothioate, phosphorodithioate, triester prodrug, sulfone,sulfonamide, sulfamate, form acetal, N-methylhydroxylamine, carbonate,carbamate, morpholino, boranophosphonate, phosphoramidate and the like.

The nucleic acid sequence included in the double-stranded siRNAaccording to the present invention can include a siRNA directed to amember of TGF-beta or other TGF-beta signaling members, and the like.

It is also possible to introduce the nucleic acid or agent according tothe present invention into phospholipid endoplasmic reticulums, such asliposome and the like, and administer the endoplasmic reticulum. Anendoplasmic reticulum in which a siRNA or shRNA is retained can beintroduced into a predetermined cell using a lipofection method. Then,the obtained cell is systemically-administered, for exampleintravenously, intra-arterially, or the like. The endoplasmic reticulumcan also be locally administered to a required site in an eye or thelike. While the siRNA exhibits an extremely excellent specificpost-transcription suppressing effect in vitro, it is quickly degradedin vivo due to nuclease activity in blood serum. Thus, the duration islimited, and because of this, there has been a need for development fora better and more effective delivery system. As to one example, Ochiya,T et al., Nature Med., 5:707-710, 1999, Curr. Gene Ther., 1: 31-52, 2001reports as follows: a biocompatible material, atelocollagen, is mixedwith a nucleic acid to form a complex, which has an action forprotecting a nucleic acid from a degrading enzyme in a living organismand which is a carrier that is extremely suitable as a carrier forsiRNA. While such a form can be used, the method for introducing anucleic acid or medicament according to the present invention is notlimited to this method. As such, due to quick degradation by the actionof the nucleic acid degrading enzyme in blood serum in a livingorganism, it becomes possible to achieve long-time continuation of theeffect. For example, Takeshita F. PNAS. (2003) 102(34) 12177-82,Minakuchi Y Nucleic Acids Research (2004) 32 (13) e109 reports asfollows: atelocollagen derived from bovine skin forms a complex with anucleic acid, which has an action for protecting a nucleic acid fromdegrading enzyme in a living organism and which is extremely suitable asa carrier of siRNA. Such a technique can be used.

As used herein, an “agent” is used in a broad sense, and may be anysubstance or other elements (e.g., energy such as light, radiation,heat, and electricity) as long as the intended objective can beattained. Examples of such a substance include, but are not limited to,proteins, polypeptides, oligopeptides, peptides, polynucleotides,oligonucleotides, nucleotides, nucleic acids (e.g., including DNA suchas cDNA and genomic DNA, and RNA such as mRNA), polysaccharides,oligosaccharides, fats, organic small molecules (e.g., hormones,ligands, information transmitting substances, organic small molecules,molecules synthesized by combinatorial chemistry, small molecules whichcan be utilized as a pharmaceutical product (e.g., a low molecularweight ligand) and the like), and composite molecule thereof.Representative examples of an agent specific to a polynucleotideinclude, but are not limited to, a polynucleotide having complementaritywith certain sequence homology (e.g., 70% or more sequence identity)relative to the sequence of the polynucleotide, a polypeptide such as atranscription factor binding to a promoter region. Representativeexamples of an agent specific to a polypeptide include, but are notlimited to, an antibody specifically directed to the polypeptide or aderivative or an analog thereof (e.g., single-stranded antibody), aspecific ligand or receptor when the polypeptide is a receptor or aligand, and a substrate when the polypeptide is an enzyme.

As used herein, “a disease, disorder, or condition associated withextracellular matrix (ECM) abnormality in a corneal endothelium” refersto those diseases, disorders, or conditions associated withextracellular matrix (ECM) abnormality of diseases, disorders, orconditions in a corneal endothelium. As such, examples can includedisorders related to Fuchs' endothelial corneal dystrophy, pterygium,allergic diseases, keratitis, corneal ulcer, and the like.

As used herein, “a disorder related to Fuchs' endothelial cornealdystrophy” refers to any disorders related to Fuchs' endothelial cornealdystrophy. Of these, disorders associated with extracellular matrix(ECM) abnormality are particularly targeted by the present invention,but it is not limited to the disorders. Examples of disorders related toFuchs' endothelial corneal dystrophy that are associated with suchextracellular matrix (ECM) abnormality include, but not limited to,photophobia, blurred vision, vision disorder, eye pain, lacrimation,hyperemia, pain, bullous keratopathy, ophthalmic unpleasantness, adecrease in contrast, glare, edema in corneal stroma, bullouskeratopathy, corneal opacity, and the like.

(General Techniques)

Molecular biological methods, biochemical methods, and microbiologicalmethods used herein are well known and commonly used in the art, whichare described in, for example, Sambrook J. et al., (1989). MolecularCloning: A Laboratory Manual, Cold Spring Harbor and the 3rd Ed. (2001);Ausubel, F. M. (1987). Current Protocols in Molecular Biology, GreenePub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide toMethods and Applications, Academic Press; Ausubel, F. M. (1992). ShortProtocols in Molecular Biology: A Compendium of Methods from CurrentProtocols in Molecular Biology, Greene Pub. Associates; Ausubel, F. M.(1995). Short Protocols in Molecular Biology: A Compendium of Methodsfrom Current Protocols in Molecular Biology, Greene Pub. Associates;Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methodsfrom Current Protocols in Molecular Biology, Wiley, and annual updates;Sninsky, J. J. et al., (1999). PCR Applications: Protocols forFunctional Genomics, Academic Press, Gait, M. J. (1985). OligonucleotideSynthesis: A Practical Approach, IRLPress; Gait, M. J. (1990).Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F.(1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press;Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids,Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistryof Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). NucleicAcids in Chemistry and Biology, Oxford University Press; Hermanson, G.T. (1996). Bioconjugate Techniques, Academic Press, ExperimentalMedicine, separate volume, “Gene Introduction & Expression AnalysisExperimental Method” Yodosha Co., Ltd., 1997, and the like. With regardto corneal endothelial cells, the report from Nancy Joyce et al.,{Joyce, 2004 #161} {Joyce, 2003 #7} is well known, while researches arecurrently conducted for effective culturing methods by conductingtransformation in a fibroblastic manner through long-term culturing andsubculturing as described above. With regard to these, the relevantportion thereof (can be the entire document) is incorporated herein byreference.

DESCRIPTION OF PREFERRED EMBODIMENT

Hereinafter, preferred embodiments will be described, but it should beunderstood that the embodiments are exemplification of the presentinvention and the scope of the present invention is not limited to suchpreferred embodiments. It should also be understood that those skilledin the art can easily perform alteration, change, and the like withinthe scope of the present invention with reference to the followingpreferable Examples.

(A Medicament for Treating or Preventing a Disease, Disorder, orCondition Associated with Extracellular Matrix (ECM) Abnormality in aCorneal Endothelium, Comprising a TGF-Beta Signal Inhibiting Agent)

In one aspect, the present invention provides medicaments for treatingor preventing a disease, disorder, or condition associated withextracellular matrix (ECM) abnormality in a corneal endothelium, themedicaments comprise a TGF-beta signal inhibiting agent. In the presentinvention, it was found that a disease, disorder, or conditionassociated with ECM in a corneal endothelium, unexpectedly ECMabnormality, could be reduced or made disappear by administering aTGF-beta signal inhibiting agent. Accordingly, it can be recognized thata use of such a TGF-beta signal inhibiting agent for the treatment orprevention of a disease, disorder, or condition associated withextracellular matrix (ECM) abnormality in a corneal endothelium couldnot be expected from previous knowledge.

In a preferable embodiment, a disease, disorder, or condition intendedby the present invention is a disorder related to Fuchs' endothelialcorneal dystrophy. For Fuchs' endothelial corneal dystrophy, currently,there is no radical therapeutic method or technique, and therapy againstFuchs' endothelial corneal dystrophy had to rely on keratoplasty. Sincethe present invention can treat extracellular matrix (ECM) abnormalitythat causes one important abnormality or disorder in Fuchs' endothelialcorneal dystrophy, it is understood to be useful in treatment orprevention of Fuchs' endothelial corneal dystrophy.

In one certain embodiment, diseases, disorders, or conditions intendedby the present invention include photophobia, blurred vision, visiondisorder, eye pain, lacrimation, hyperemia, pain, bullous keratopathy,ophthalmic unpleasantness, a decrease in contrast, glare, edema incorneal stroma, and corneal opacity in Fuchs' endothelial cornealdystrophy.

The subject of the administration (transplantation) of the medicament ormethod according to the present invention includes mammals (e.g.,humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep,monkeys, and the like), and the subject is preferably primates, andparticularly preferably humans. Therapy for corneal endothelium inprimates had not achieved sufficient results before, and from that pointof view, the present invention provides an innovative therapeutic methodand medicament.

TGF-beta signal transduction pathways are broadly classified into theSmad2/3 system through ALK4, 5, or 7 and the Smad1/5/8 system throughALK1, 2, 3, or 6. Both of them are well known to be related to fibrosis(J. Massagu'e, Annu. Rev. Biochem. 1998.67:753-91; Vilar J M G, JansenR, Sander C (2006) PLoS Comput Biol 2(1):e3; Leask, A., Abraham, D. J.FASEB J. 18, 816-827 (2004); Coert Margadant & Arnoud Sonnenberg EMBOreports (2010) 11, 97-105; Joel Rosenbloom et al., Ann Intern Med. 2010;152:159-166.). It is also known that BMP-7 can suppress a TGF-betasignal to suppress fibrosis (other than the above-described literatures,Ralf Weiskirchen, et al., Frontiers in Bioscience 14, 4992-5012, Jun. 1,2009; Elisabeth M Zeisberg et al., Nature Medicine 13, 952-961 (2007);Michael Zeisberg et al., Nature Medicine 9, 964-968 (2003)). However,these literatures describe involvement with TGF-beta with regard toluetic interstitial keratitis, which is a very special disease, or sucha state as to accompany a membrane-shaped tissue actually consisting ofan extracellular substrate, such as collagen and the like, by a severedisorder made artificially. However, it is difficult to expect atherapeutic effect from this description. In addition, those literaturesshow that fibrosis at the time of a corneal severe disorder is caused byIL-1beta, or by activation of p38 MAPK halfway, while those show, usinga rabbit, that fibrosis seen when severe inflammation in a living bodyoccurs due to excess freezing trauma in a rabbit accompanies activationof p38 MAPK, and an inhibiting agent can partly suppress fibrosis. Thesepieces of knowledge show that activation of p38 MAPK is accompanied insuch a condition that very strong inflammation occurs in a living bodyand a membrane-shaped tissue consisting of an extracellular substrate isaccompanied. Those do not mention that a TGF-beta signal inhibitingagent is effective to treat or prevent a disease, disorder, or conditionassociated with extracellular matrix (ECM) abnormality in a cornealendothelium of Fuchs' endothelial corneal dystrophy and the like. Thosesuggest nothing about maintenance of a normal condition. As describedabove, it was previously believed difficult to culture a cornealendothelial cell with keeping the normal functions. In the previousreports, finally, a disease, disorder, or condition associated withextracellular matrix (ECM) abnormality in a corneal endothelium ofFuchs' endothelial corneal dystrophy and the like could not be treatedor prevented, let alone it was not believed possible to treat or preventa disease, disorder, or condition associated with extracellular matrix(ECM) abnormality in a corneal endothelium of Fuchs' endothelial cornealdystrophy and the like by suppressing TGF-beta signal transductionpathways.

The TGF-beta signal inhibiting agent used in the present invention maybe any agent as long as the agent can inhibit the signal pathway ofTGF-beta. In addition, the TGF-beta signal transduction pathway to beinhibited may be associated with a factor associated with any signal, aslong as such a signal transduction pathway ultimately exerts an effectsimilar (opposite in a case of an inhibiting agent, an antagonist, orthe like) to the signal transduction pathway of TGF-beta, like BMP-7, inaddition to signal transduction pathways with which TGF-beta and aTGF-beta receptor are directly associated, as is well known.

In the present invention, it is possible to include a TGF-beta signalinhibiting agent alone, and it is also possible to include several typesthereof in combination with each other as needed.

In one embodiment, the TGF-beta signal inhibiting agent includes atleast one of an antagonist of TGF-beta, an antagonist of a receptor ofTGF-beta, or an inhibiting agent of Smad3, ingredients illustrated inother parts of the present specification, a pharmaceutically acceptablesalt or a solvate thereof, or a solvate of the pharmaceuticallyacceptable salt. As for the antagonist of TGF-beta, the antagonist of areceptor of TGF-beta, and the inhibiting agent of Smad3, any one of themdescribed in other parts of the present specification can be used.

In one embodiment, TGF-beta signal inhibiting agents that may be used inthe present invention include at least one of SB431542(4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide),BMP-7, anti-TGF-beta antibody, anti-TGF-beta receptor antibody, siRNA ofTGF-beta, siRNA of a TGF-beta receptor, an antisense oligonucleotide ofTGF-beta,6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone,A83-01(3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide),Stemolecule™ TLK inhibitor(2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine),Stemolecule™ BMP inhibitor LDN-193189(6-(4-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine),SD-208 (2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine),LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), componentsillustrated in other parts of the present specification,pharmaceutically acceptable salts or solvates thereof, or solvates ofthe pharmaceutically acceptable salts. It should be noted that theabove-mentioned antibodies may be, but not limited to, neutralizingantibodies. Without wishing to be bound by any theory, an effect oftreating or preventing a disease, disorder, or condition associated withextracellular matrix (ECM) abnormality in a corneal endothelium ofFuchs' endothelial corneal dystrophy and the like is observed for bothof SB431542 that attains an effect through Smad2/3 (related to ALK4, 5,and 7), and BMP-7 that attains an effect through Smad1/5/8 (related toALK1, 2, 3, and 6). Accordingly, it is understood that even if they areTGF-beta signal inhibiting agents for either of these pathways, theeffect of the present invention can be achieved.

In a preferable embodiment, TGF-beta signal inhibiting agents used inthe present invention include SB431542 (4-[4-(1,3-benzodioxol-5-yl)2-pyridinyl)-1H-imidazol-2-yl]benzamide). The reasonis that improvement of a disease, disorder, or condition associated withextracellular matrix (ECM) abnormality in a corneal endothelium ofFuchs' endothelial corneal dystrophy and the like was exhibited. In apreferred embodiment, SB431542 is included to be present at aconcentration of about 0.1 μM to about 10 μM in use, preferably includedto be present at a concentration of about 1 μM to about 10 μM in use,and further preferably included to be present at a concentration ofabout 1 μM in use.

The concentration of the TGF-beta signal inhibiting agent used in thepresent invention is normally about 0.1 to 100 μmol/l, preferably about0.1 to 30 μmol/l, and more preferably about 1 μmol/l; when several typesthereof are used, the concentration may be changed appropriately, andexamples of other concentration ranges are normally about 0.001 to 100μmol/l, preferably, about 0.01 to 75 μmol/l, about 0.05 to 50 μmol/l,about 1 to 10 μmol/l, about 0.01 to 10 μmol/l, about 0.05 to 10 μmol/l,about 0.075 to mol/l, about 0.1 to 10 μmol/l, about 0.5 to 10 μmol/l,about 0.75 to 10 μmol/l, about 1.0 to 10 μmol/l, about 1.25 to 10μmol/l, about 1.5 to 10 μmol/l, about 1.75 to 10 mol/l, about 2.0 to 10μmol/l, about 2.5 to 10 μmol/l, about 3.0 to 10 μmol/l, about 4.0 to 10μmol/l, about 5.0 to 10 μmol/l, about 6.0 to 10 μmol/l, about 7.0 to 10μmol/l, about 8.0 to 10 μmol/l, about 9.0 to 10 μmol/l, about 0.01 to 50μmol/l, about 0.05 to 5.0 μmol/l, about 0.075 to 5.0 μmol/l, about 0.1to 5.0 μmol/l, about 0.5 to 5.0 μmol/l, about 0.75 to 5.0 μmol/l, about1.0 to 5.0 μmol/l, about 1.25 to 5.0 μmol/l, about 1.5 to 5.0 μmol/l,about 1.75 to 5.0 μmol/l, about 2.0 to 5.0 μmol/l, about 2.5 to 5.0μmol/l, about 3.0 to 5.0 μmol/l, about 4.0 to 5.0 μmol/l, about 0.01 to3.0 μmol/l, about 0.05 to 3.0 μmol/l, about 0.075 to 3.0 μmol/l, about0.1 to 3.0 μmol/l, about 0.5 to 3.0 μmol/l, about 0.75 to 3.0 μmol/l,about 1.0 to 3.0 μmol/l, about 1.25 to 3.0 μmol/l, about 1.5 to 3.0μmol/l, about 1.75 to 3.0 μmol/l, about 2.0 to 3.0 μmol/l, about 0.01 to1.0 μmol/l, about 0.05 to 1.0 μmol/l, about 0.075 to 1.0 μmol/l, about0.1 to 1.0 μmol/l, about 0.5 to 1.0 μmol/l, about 0.75 to 1.0 μmol/l,about 0.09 to 35 μmol/l, and about 0.09 to 3.2 mol/l, and morepreferably can include, but not limited to, about 0.05 to 1.0 μmol/l,about 0.075 to 1.0 μmol/l, about 0.1 to 1.0 μmol/l, about 0.5 to 1.0μmol/l, and about 0.75 to 1.0 μmol/1.

In a preferable embodiment, a TGF-beta signal inhibiting agent usedincludes4-[4-(1,3-benzodioxol-5-yl)2-pyridinyl)-1H-imidazol-2-yl]benzamide or apharmaceutically acceptable salt thereof.

In another preferred embodiment, the TGF-beta signal inhibiting agentused in the present invention includes BMP-7. This is because fibrosiswas suppressed, and moreover, it was indicated that the protein incharge of the normal functions was retained, and transplant to primateswas bearable. In a preferred embodiment, BMP-7 is included to be presentat a concentration of about 10 ng/ml to about 1,000 ng/ml in use, andmore preferably, included to be present at a concentration of about 100ng/ml to about 1,000 ng/ml in use. BMP-7 may be included to be presentat a concentration of about 100 ng/ml in use, or may be included to bepresent at a concentration of about 1,000 ng/ml.

A medicament for treating or preventing according to the presentinvention may comprise an additional medicinal component. Representativeexamples of such medicinal products include Rho kinase inhibiting agentsand steroids. Without wishing to be bound by any theory, it is becauseinclusion of a Rho kinase inhibiting agent promotes adhesion of acorneal endothelial cell to prevent the cell from shedding, it enablesthe formation of a corneal endothelial cell layer having good cellmorphology and high cell density, and thereby an effect of a TGF-βsignal inhibiting agent can be enhanced. In the present invention, onetype of Rho kinase inhibiting agent can be included alone, or severaltypes thereof can be used in combination and included as necessary.

Examples of Rho kinase inhibiting agents that may be used in the presentinvention include compounds disclosed in the following documents: U.S.Pat. No. 4,678,783, Japanese Patent No. 3421217, InternationalPublication No. WO 95/28387, International Publication No. WO 99/20620,International Publication No. WO 99/61403, International Publication No.WO 02/076976, International Publication No. WO 02/076977, InternationalPublication No. WO 2002/083175, International Publication No. WO02/100833, International Publication No. WO 03/059913, InternationalPublication No. WO 03/062227, International Publication No. WO2004/009555, International Publication No. WO 2004/022541, InternationalPublication No. WO 2004/108724, International Publication No. WO2005/003101, International Publication No. WO 2005/039564, InternationalPublication No. WO 2005/034866, International Publication No. WO2005/037197, International Publication No. WO 2005/037198, InternationalPublication No. WO 2005/035501, International Publication No. WO2005/035503, International Publication No. WO 2005/035506, InternationalPublication No. WO 2005/080394, International Publication No. WO2005/103050, International Publication No. WO 2006/057270, InternationalPublication No. WO 2007/026664 and the like. Such compounds each can bemanufactured by the methods described in the documents in which therespective compounds are disclosed. The specific examples can include1-(5-isoquinolinesulfonyl)homopiperazine or a salt thereof (e.g.,Fasudil (1-(5-isoquinolinesulfonyl)homopiperazine)),(R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide or asalt thereof (e.g., Y-27632((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamidedihydrochloride monohydrate) and the like) and the like.

The concentration of the Rho kinase inhibiting agent in the presentinvention is normally about 1 to 100 μmol/l, preferably, about 5 to 20μmol/l, and more preferably about 10 μmol/l; when several types thereofare used, the concentration may be changed appropriately, and examplesof other concentration ranges are normally about 0.001 to 100 μmol/l,preferably, about 0.01 to 75 μmol/l, about 0.05 to 50 μmol/l, about 1 to10 mol/l, about 0.01 to 10 μmol/l, about 0.05 to 10 μmol/l, about 0.075to 10 μmol/l, about 0.1 to 10 μmol/l, about 0.5 to 10 μmol/l, about 0.75to 10 μmol/l, about 1.0 to 10 mol/l, about 1.25 to 10 μmol/l, about 1.5to 10 μmol/l, about 1.75 to 10 μmol/l, about 2.0 to 10 μmol/l, about 2.5to 10 μmol/l, about 3.0 to 10 μmol/l, about 4.0 to 10 μmol/l, about 5.0to 10 μmol/l, about 6.0 to 10 μmol/l, about 7.0 to 10 μmol/l, about 8.0to 10 mol/l, about 9.0 to 10 μmol/l, about 0.01 to 50 μmol/l, about 0.05to 5.0 μmol/l, about 0.075 to 5.0 μmol/l, about 0.1 to 5.0 μmol/l, about0.5 to 5.0 μmol/l, about 0.75 to 5.0 mol/l, about 1.0 to 5.0 μmol/l,about 1.25 to 5.0 μmol/l, about 1.5 to 5.0 μmol/l, about 1.75 to 5.0μmol/l, about 2.0 to 5.0 μmol/l, about 2.5 to 5.0 μmol/l, about 3.0 to5.0 mol/l, about 4.0 to 5.0 μmol/l, about 0.01 to 3.0 μmol/l, about 0.05to 3.0 μmol/l, about 0.075 to 3.0 μmol/l, about 0.1 to 3.0 μmol/l, about0.5 to 3.0 μmol/l, about 0.75 to 3.0 mol/l, about 1.0 to 3.0 μmol/l,about 1.25 to 3.0 μmol/l, about 1.5 to 3.0 μmol/l, about 1.75 to 3.0μmol/l, about 2.0 to 3.0 μmol/l, about 0.01 to 1.0 μmol/l, about 0.05 to1.0 mol/l, about 0.075 to 1.0 μmol/l, about 0.1 to 1.0 μmol/l, about 0.5to 1.0 μmol/l, about 0.75 to 1.0 μmol/l, about 0.09 to 35 μmol/l, andabout 0.09 to 3.2 μmol/l, and more preferably can include, but notlimited to about 0.05 to 1.0 μmol/l, about 0.075 to 1.0 mol/l, about 0.1to 1.0 μmol/l, about 0.5 to 1.0 μmol/l, and about 0.75 to 1.0 μmol/1.

The present invention can be administered as eye-drops.

The dosage amount and the frequency of administration vary in accordancewith symptoms, ages, weights or administration forms. In case of the useas an eye lotion, for example, for normal adults, the formulation,containing an effective ingredient of about 0.0001 to 0.1 w/v %, andpreferably about 0.003 to 0.03 w/v %, can be administered 1 to 10 timesper day, preferably 1 to 6 times per day, and more preferably 1 to 3times per day, and at the amount in the range of about 0.01 to 0.1 mLper time. When the medicament according to the present invention isintroduced into an anterior chamber, the medicament at a concentrationone-tenth to one-thousandth of the above-mentioned concentration can beused. Those skilled in the art can appropriately select the type andconcentration of TGF-beta signal inhibiting agent, Rho kinase inhibitingagent, and the like in accordance with disease states.

In another aspect, the present invention provides a TGF-beta signalinhibitory substance for treatment or prevention of a disorderassociated with extracellular matrix (ECM) abnormality in a cornealendothelium. A TGF-beta signal inhibitory substance can be usedinterchangeably with a TGF-beta signal inhibiting agent. In this use,with regard to the extracellular matrix (ECM) abnormality in a cornealendothelium and the TGF-beta signal inhibiting agent, any embodimentsdescribed herein can be used.

In another aspect, the present invention provides a method for treatingor preventing a disorder associated with extracellular matrix (ECM)abnormality in a corneal endothelium in a subject, wherein the methodcomprises a step of administering an effective amount of a TGF-betasignal inhibiting agent to the subject. In this method, with regard tothe extracellular matrix (ECM) abnormality in a corneal endothelium andthe TGF-beta signal inhibiting agent, any embodiments described hereincan be used.

The subject of the administration (transplantation) of the medicament ormethod according to the present invention includes mammals (e.g.,humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep,monkeys, and the like), and the subject is preferably primates, andparticularly preferably humans. Therapy for corneal endothelium inprimates had not achieved sufficient results before, and from that pointof view, the present invention provides an innovative therapeutic methodand medicament.

Reference literatures such as scientific literatures, patents, andpatent applications cited herein are incorporated herein by reference tothe same extent that the entirety of each document is specificallydescribed.

As described above, the present invention has been illustrated byshowing preferable embodiments to facilitate understanding. The presentinvention is illustrated below based on Examples. The aforementionedillustration and the following Examples are not provided for the purposeof limiting the present invention, but for the sole purpose ofexemplification. Thus, the scope of the present invention is not limitedto embodiments and Examples specifically described herein and is limitedonly by the scope of claims.

EXAMPLES

Hereinafter, examples of normally culturing a cell of a cornealendothelial cell according to the present invention will be described.In applicable, standards stipulated by Ministry of Health, Labour andWelfare, Ministry of Education, Culture, Sports, Science and Technology,or the like were recognized for the handling of biological samples orthe like; and if applicable, the handling was performed based onHelsinki Declaration or ethical codes prepared based on the Declaration.For the donation of eyes used for the research, agreements were obtainedfrom close relatives of all the deceased donors. The present researchwas approved by the institutional review board of Erlangen university(Doyle), SightLife™ (Seattle, WA) eye bank, or those in accordancetherewith.

In Fuchs' endothelial corneal dystrophy, corneal endothelial cells leadto cell death, and when remaining corneal endothelial cells cannotcompensate a pumping function and a barrier function, then thetransparency of the cornea cannot be maintained, leading to loss ofsight due to corneal opacity. In addition, it is known that a cornealendothelial cell of a Fuchs' endothelial corneal dystrophy patientoverproduces an extracellular matrix to cause guttae formation andthickening of Descemet's membrane. Since the guttae formation and thethickening of Descemet's membrane cause light scattering and the like,it causes reduced visual acuity, photophobia, blurred vision tosignificantly injure the QOL of the patients. An immobilized cornealendothelial cell strain (iFECD) derived from Fuchs' endothelial cornealdystrophy patients was used as a model, and compared with an immobilizedcorneal endothelial cell strain (iHCEC) derived from healthy donors toclarify a cause relating to production of an extracellular matrix, andidentify a therapy target.

(Preparation Example: Production of an Immobilized Corneal EndothelialCell Strain (iFECD) Model Derived from a Fuchs' Endothelial CornealDystrophy Patient)

In the present example, immobilized corneal endothelial cell strains(iFECD) from corneal endothelial cells derived from a Fuchs' endothelialcorneal dystrophy patient were produced.

(Culture Method)

Corneal endothelial cells were mechanically peeled off with a basalmembrane from a corneal for research purchased from the Seattle EyeBank. After collagenase was used to detach and collect the cornealendothelial cell from the basal membrane, the cells were subjected toprimary culture. With regard to a medium, Opti-MEM I Reduced-SerumMedium, Liquid (INVITROGEN catalog No.: 31985-070) to which 8% FBS(BIOWEST, catalog No.: S1820-500), 200 mg/ml CaCl₂)·2H₂O (SIGMA catalogNo.: C7902-500G), 0.08% chondroitin sulfate (SIGMA catalog No.:C9819-5G), 20 μg/ml ascorbic acid (SIGMA catalog No.: A4544-25G), 50μg/ml gentamicin (INVITROGEN catalog No.: 15710-064) and 5 ng/ml EGF(INVITROGEN catalog No.: PHG0311) were added and that was acclimated fora 3T3 feeder cell, was used as a basal medium. Further, the cells werecultured in a basal medium to which SB431542 (1 μmol/l) and SB203580(4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5(4-pyridyl)imidazole<4-[4-(4-fluorphenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine)(1 μmol/l) were added (referred to as “SB203580+SB431542+3T3 acclimatedmedium”).

(Method of Acquisition)

Corneal endothelial cells from three human patients that led to bullouskeratopathy by clinical diagnosis of Fuchs' endothelial cornealdystrophy and underwent corneal endothelial transplantation (Descemetmembrane endothelial keratoplasty=DMEK) were obtained under the writtenconsent and the approval of Ethics Committee. In DMEK, mechanicallypathological corneal endothelial cells and Descemet membrane, which is abasement membrane, were exfoliated together and immersed in Optisol-GS(Bausch & Lomb Incorporated), which is a corneal storage solution. Afterthat, collagenase treatment was carried out to enzymatically collectcorneal endothelial cells, which was then cultured inSB203580+SB431542+3T3-conditioned medium. With regard to the culturedcorneal endothelial cells derived from Fuchs' endothelial cornealdystrophy patients, SV40 large T antigen and hTERT genes were amplifiedby PCR and introduced into a lentiviral vector (pLenti6.3_V5-TOPO; LifeTechnologies Inc.). The lentiviral vector was then used to infect 293Tcells (RCB2202; Riken Bioresource Center, Ibaraki, Japan) by atransfection reagent (Fugene HD; Promega Corp., Madison, WI), togetherwith three types of helper plasmids (pLP1, pLP2, pLP/VSVG; LifeTechnologies Inc.). Culture supernatant comprising viruses was collectedafter 48 hours from the infection. It is added using 5 μg/ml polybreneto culture solutions of cultured corneal endothelial cells derived fromFuchs' endothelial corneal dystrophy patients to introduce SV40 large Tantigen and hTERT genes. Phase-contrast microscope images of animmobilized corneal endothelial cell strain (iFECD) derived from Fuchs'endothelial corneal dystrophy patients were confirmed. Cornealendothelial cells cultured from a cornea for research imported as acontrol from a Seattle eye bank were immobilized by a similar method tomake an immobilized cell strain (iHCEC) of normal corneal endothelialcell. When seeing phase-contrast microscope images of an immobilizedcorneal endothelial cell strain (iHCEC) derived from healthy donors, andan immobilized corneal endothelial cell strain (iFECD), both of theiHCEC and the iFECD have the same one-layered polygonal form as a normalcorneal endothelial cell. In the cases of the iHCEC and the iFECD,SB431542 of which maintenance culture was carried out in DMEM+10% FBSwas obtained from TOCRIS Corporation (Catalog number: 1614). SB203580was obtained from CALBIOCHEM (Catalog number: 559389).

Preparation Example 2: Confirmation of the Normal Functions of anImmobilized Corneal Endothelial Cell Strain (iFECD)

In the present example, the normal function of an immobilized cornealendothelial cell strain (iFECD) was confirmed.

(Immunostaining with Na⁺/K⁺-ATPase and ZO-1)

At first, in order to confirm the normal functions of an immobilizedcorneal endothelial cell strain (iFECD), immunostaining withNa⁺/K⁺-ATPase and ZO-1 was carried out. It is to confirm a pumpingfunction and a barrier function, which are functions of a cornealendothelial cell. Na⁺/K⁺-ATPase and ZO-1 each exhibit the normality of apumping function and a barrier function, which are functions of acorneal endothelial cell. A technique is as follows.

(Cell Observation Method Including Staining and the Like (HistologicalTest))

Cells were observed under a phase-contrast microscope. In addition,after cells were fixed, ZO-1 and Na⁺/K⁺-ATPase were used asfunction-related markers to carry out immunostaining and observationunder a fluorescence microscope. For a tissue staining test, culturedcells are put in Lab-Tek™ Chamber Slides' (NUNC A/S, Roskilde, Denmark),and then fixed at room temperature (RT) for 10 minutes in 4%formaldehyde, and incubated with 1% bovine serum albumin (BSA) for 30minutes. Specifically, cultured cells on Lab-Tek™ Chamber Slides™ (NUNCA/S, Roskilde, Denmark) are fixed at room temperature for 10 minutes in4% formaldehyde, and then incubated with 1% bovine serum albumin (BSA)for 30 minutes. In order to examine the expression type of the cells,ZO-1 (Zymed Laboratories, Inc., South San Francisco, CA), which is atight junction-related protein, and Na⁺/K⁺-ATPase (Upstate Biotec, Inc.,Lake Placid, NY), which is a pumping function-related protein, wereimmunohistochemically analyzed. ZO-1 and Na⁺/K⁺-ATPase were used asmarkers relating to cell functions. Staining of ZO-1 and Na⁺/K⁺-ATPasewere carried out using 1:200 dilutions of a ZO-1 polyclonal antibody anda Na⁺/K⁺-ATPase monoclonal antibody, respectively. For a secondaryantibody, 1:2000 dilution of Alexa Fluor (registered trademark) 488labeled, or Alexa Fluor (registered trademark) 594 labeled goatanti-mouse IgG (Life Technologies) was used. Cell nuclei were thenstained with DAPI (Vector Laboratories, Inc., Burlingame, CA) or PI(Sigma-Aldrich). A slide was then observed under a fluorescencemicroscope (TCS SP2 AOBS; Leica Microsystems, Welzlar, Germany).

When seeing the result, both of the iHCEC and the iFECD expressedNa⁺/K⁺-ATPase and ZO-1 in all the cells, and it was shown that theimmobilized cell strain made maintained the normal functions.

In addition, morphological observation images of the iHCEC and the iFECDunder a transmission electron microscope are shown. The iHCEC and theiFECD were cultured in DMEM on Transwell without serum. One week later,they were fixed in a confluent state and when the morphology wasobserved under a transmission electron microscope, they were shown to beone-layered cells that are not recognized to have morphologicallyapparent abnormality.

In addition, it is known that a corneal endothelial cell of a Fuchs'endothelial corneal dystrophy patient overproduces an extracellularmatrix to cause guttae formation and thickening of Descemet's membrane.Accordingly, with regard to expression of collagen type I, collagen typeIV, and Fibronectin, which are proteins constituting an extracellularmatrix, the iHCEC and the iFECD were cultured on culture dishes andimmunostained. In the iFECD, it was shown that expression of collagentype I, collagen type IV, and Fibronectin was increased in comparisonwith the iHCEC. In addition, when the gene expression level of thecultured iHCEC and iFECD was examined by a real-time PCR method, it wasrecognized that collagen type I and Fibronectin significantly promotedthe expression level, and that collagen type IV tended to promote theexpression.

It was examined whether the iFECD overproduces an extracellular matrixin the same way as a corneal endothelium of Fuchs' endothelial cornealdystrophy patients. The iHCEC and the iFECD were cultured in DMEM onTranswell without serum, one week after which they were fixed in aconfluent state and HE-stained. In the iFECD, it was recognized that asignificantly thickened extracellular matrix was produced in comparisonwith the iHCEC. As described above, a disease model cell was made whichhas a characteristic of overproduction of an extracellular matrix inFuchs' endothelial corneal dystrophy patients. Since analysis using thedisease model cell is expected to contribute to clarifying thepathological condition of Fuchs' endothelial corneal dystrophy, forwhich there are many unclear points, this cell was used to try thedevelopment of a therapeutic medicament for Fuchs' endothelial cornealdystrophy in the following.

Example 1: Real-Time PCR Analysis of the Expression Amount of a GeneRelated to Epithelial-Mesenchymal Transition (EMT) Related to Productionof an Extracellular Matrix

With regard to the iHCEC and the iFECD, the present example shows aresult of analyzing, by real-time PCR, the expression amount of a generelated to epithelial-mesenchymal transition (EMT) related to productionof an extracellular matrix.

(Real-Time PCR)

-   -   Real-time PCR method: In addition, PCR was performed by a Taqman        method for Snail1, Snail2, or ZEB1 in accordance with the        following method. A Taqman probe was purchased from INVITROGEN.        The mRNA amount of collagen type I, collagen type IV, and        Fibronectin was examined by a real-time PCR method. RNEasy        (QIAGEN, Catalog number: 74106) was used in extraction of the        total RNA from cells. The extracted RNA was subjected to a        reverse transcription reaction (42° C., 60 minutes) with        ReverTra Ace (TOYOBO Co., Ltd., Catalog number: TRT-101), and        collagen type I, collagen type IV, and Fibronectin were        amplified with a reaction reagent TaqMan Fast Advanced mastermix        (Applied Biosystems) using GAPDH as an internal standard. Probes        (a labeled primer set available from Applied Biosystems)        described below was used in the PCR reaction, which was        performed in the StepOne™ (Applied Biosystems) real-time PCR        system.    -   Snail1 Hs00195591_ml SNAI1    -   Snail2 Hs00950344_ml SNAI2    -   ZEB1 Hs00232783_ml ZEB1    -   GAPDH TaqMan® pre developed Assay Reagents Human GADPH (cat no.:        4333764F).

(Result)

The result is shown in FIG. 1 . As shown in FIG. 1 , when the expressionamount of a gene related to epithelial-mesenchymal transition (EMT)related to production of an extracellular matrix was analyzed byreal-time PCR, it was recognized in Snail1 and ZEB1 that the iFECDsignificantly promoted the expression in comparison with the iHCEC.

(Promotion of Expression of Snail1 and ZEB1 by the TGF-Beta)

In order to confirm whether expression promotion of Snail1 and ZEB1relates to production of an extracellular matrix, stimulation withTGF-beta, which is known to accelerate expression of Snail1 and ZEB1,was performed. The technique is as described below. The iFECD and theiHCEC were cultured in DMEM containing 10% fetal bovine serum, andcultured overnight in DMEM without 10% fetal bovine serum. After that,expression of Snail1, ZEB1, collagen type I, collagen type IV, collagentype VIII, Fibronectin was examined by a real-time PCR method. Probesdescribed below were used in the PCR reaction, which was performed inthe StepOne™ (Applied Biosystems) real-time PCR system.

-   -   Snail1 Hs00195591_ml SNAI1    -   ZEB1 Hs00232783_ml ZEB1    -   collagen type I Hs00164004_ml COL1A1    -   collagen type IV Hs00266327_ml COL4A1    -   collagen type VIII Hs00697025_ml COL8A2    -   Fibronectin Hs01549976_ml FN1    -   GAPDH TaqMan® pre developed Assay Reagents Human GADPH (cat no.:        4333764F).

The result is shown in FIG. 2 . It was confirmed that TGF-betasignificantly accelerates expression of Snail1 and ZEB1 in the iFECD (A,B). Accordingly, when the gene expression amount of a proteinconstituting an extracellular matrix was analyzed by real-time PCR,expression of collagen type I, collagen type IV, collagen type VIII, andFibronectin was significantly accelerated.

Example 2: Acceleration of an iFECD-Produced Extracellular Matrix byTGF-Beta

In the present example, it was examined whether an iFECD-producedextracellular matrix was accelerated by TGF-beta.

The iHCEC and the iFECD were cultured in DMEM on Transwell withoutserum, one week after which they were fixed in a confluent state andHE-stained. The procedure is as described below. If required,deparaffinization (for example, with pure ethanol) and water washingwere carried out, and the sample was immersed in hematoxylin from Omnifor 10 minutes. Thereafter, water washing with running water wasperformed, and ammonia water was used for developing color for 30seconds. Thereafter, water washing with running water for 5 minutes,staining for 2 minutes with a ten-fold diluted solution of eosinhydrochloride, dehydration, lucidification, and mounting were performed.It was recognized in the iHCEC and the iFECD that TGF-beta stimulationproduced a significantly thickened extracellular matrix. Further, it wasrecognized that in the presence of TGF-beta, the iFECD produced asignificantly thickened extracellular matrix in comparison with theiHCEC.

These show that in corneal endothelial cells of Fuchs' endothelialcorneal dystrophy patients, the expression level of Snail1 and ZEB1 ishigh and that the production quantity of an extracellular matrix inresponse to the TGF-beta stimulation is significantly higher thancorneal endothelial cells of a healthy subject.

Example 3: An Effect on the Extracellular Matrix Production bySuppression of Snail1 and ZEB1 Using siRNA

In the present example, an effect on the extracellular matrix productionby suppression of Snail1 and ZEB1 using siRNA was examined in order todemonstrate that the expression promotion of Snail1 and ZEB1 causesproduction of an extracellular matrix. The experimental procedure is asdescribed below.

(Technique)

The iFECD and the iHCEC were seeded and incubated with Snail1 StealthRNAi™ (Life Technologies Corp., Carlsbad, CA) or ZEB1 Stealth RNAi™(Life Technologies Corp., Carlsbad, CA) and Lipofectamine™ RNAiMAX (LifeTechnologies Corp., Carlsbad, CA) at 37° C. for 12 hours. A randomsequence of RNAi was used as a control. Thereafter, cells are passagedand used in the experiment. Three types of each of Snail1 Stealth RNAi™and ZEB1 Stealth RNAi™ were used to carry out the experiment, andrepresentative examples were shown as the results. Cells in which Snail1or ZEB1 was knocked down by siRNA were seeded, and expression of Snail1,ZEB1, collagen type I, collagen type IV, collagen type VIII, andFibronectin was examined by a real-time PCR method. Probes describedbelow were used in the PCR reaction, which was performed in the StepOne™(Applied Biosystems) real-time PCR system.

(Material)

siRNA

siRNA of Snail1 (SNAI1 HSS143995*, SNAI1 HSS143996, SNAI1 HSS143997)

siRNA of ZEB1 (ZEB1 HSS110548*, ZEB1 HSS110549, ZEB1 HSS186235) Itshould be noted that siRNA shown in the result was described as *.

Probes in a Real-Time PCR Method

-   -   Snail1 Hs00195591_ml SNAI1    -   ZEB1 Hs00232783_ml ZEB1    -   Collagen type I Hs00164004_ml COL1A1    -   Collagen type IV Hs00266327_ml COL4A1    -   Collagen type VIII Hs00697025_ml COL8A2    -   Fibronectin Hs01549976_ml FN1    -   GAPDH TaqMan® pre developed Assay Reagents Human GADPH (cat no.:        4333764F).

(Result)

The result is shown in FIG. 4 . It was confirmed that siRNA suppressesexpression of Snail1 and ZEB1 (A, F). The expression suppression ofSnail1 or ZEB1 by siRNA significantly suppressed expression of collagentype I, collagen type IV, collagen type VIII, and Fibronectin. From thisresult, it was found that ZEB1 or Snail1 negatively controls geneexpression of an extracellular-constituting protein.

(Adjustment of Expression of Collagen Type I, Collagen Type IV, andFibronectin by Immunostaining)

It was then confirmed by immunostaining that expression of collagen typeI, collagen type IV, and Fibronectin was suppressed. The technique ofimmunostaining is similar to the above-described Preparation Example 2.With regard to an antibody, antibodies against collagen type I, collagentype IV, and Fibronectin were used instead to carry out the experiment.

-   -   An antibody against collagen type I: Anti collagen type I        (Rabbit polyclonal) (ROCKLLAND™ antibodies and assays, Cat no.:        600-401-103S)    -   An antibody against collagen type IV: collagen type IV (Rabbit        polyclonal) (Abcam, Cat no.: ab6586)    -   An antibody against Fibronectin: Anti-fibronectin (mouse        monoclonal) (BD Biosciences, Cat no.: 610077)

For a tissue staining test, cultured cells were put in Lab-Tek™ ChamberSlides™ (NUNC A/S, Roskilde, Denmark), fixed with 4% formaldehyde for 10minutes at room temperature (RT), and incubated with 1% bovine serumalbumin (BSA) for 30 minutes. Specifically, cultured cells on Lab-Tek™Chamber Slides™ (NUNC A/S, Roskilde, Denmark) were fixed at roomtemperature for 10 minutes in 4% formaldehyde, and then incubated with1% bovine serum albumin (BSA) for 30 minutes. In order to examineexpression of an extracellular matrix produced by cells, a 1:200dilution of each of antibodies against collagen type I, collagen typeIV, and Fibronectin was used to carry out. For a secondary antibody, a1:2000 dilution of Alexa Fluor (registered trademark) 488 labeled, orAlexa Fluor (registered trademark) 594 labeled goat anti-mouse IgG (LifeTechnologies) was used. Cell nuclei were then stained with DAPI (VectorLaboratories, Inc., Burlingame, CA) or PI (Sigma-Aldrich). A slide wasthen observed under a fluorescence microscope (TCS SP2 AOBS; LeicaMicrosystems, Welzlar, Germany).

(Result)

The result is shown in FIG. 5 . As shown in FIG. 5 , it was alsoconfirmed that expression suppression of Snail1 or ZEB1 by siRNA alsosuppressed expression of collagen type I, collagen type IV, collagentype VIII, and Fibronectin at a protein level.

Example 4: Suppression of Overproduction of an Extracellular Matrix inthe iFECD by Expression Suppression of Snail1 or ZEB1

In the present example, it was confirmed that expression suppression ofSnail1 or ZEB1 suppressed overproduction of an extracellular matrix inthe iFECD.

Further, the iHCEC and the iFECD were cultured in DMEM on Transwellwithout serum, one week after which they were fixed in a confluent stateand HE-stained. The HE-staining was carried out in accordance with theprocedure of the above-described examples.

(Result)

The result is shown in FIG. 6 . As shown in FIG. 6 , expressionsuppression of Snail1 or ZEB1 by siRNA suppressed overproduction of anextracellular matrix in the iFECD to result in the normal level.Accordingly, it was found that suppression of ZEB1 or Snail can suppressoverproduction of an extracellular matrix in a Fuchs' endothelialcorneal dystrophy cell.

Example 5: Adjustment of Extracellular Matrix Abnormality in a CornealEndothelium by a TGF-Beta Signal Inhibiting Agent

It was then examined whether a TGF-beta signal inhibiting agent,SB431542, can be used to inhibit a TGF-beta signal and adjustextracellular matrix abnormality in a corneal endothelium. SB431542 wasobtained from TOCRIS Corporation (Catalog number: 1614).

(Examination by Real-Time PCR)

The gene expression amount was confirmed by real-time PCR. Real-time PCRwas carried out in accordance with the above-described examples. Thefollowing probes were used for collagen type I, collagen type IV,collagen type VIII, and Fibronectin.

-   -   Collagen type I Hs00164004_ml COL1A1    -   Collagen type IV Hs00266327_ml COL4A1    -   Collagen type VIII Hs00697025_ml COL8A2    -   Fibronectin Hs01549976_ml FN1    -   GAPDH TaqMan® pre developed Assay Reagents Human GADPH (cat no.:        4333764F)

(Result)

The result is shown in FIG. 7 . As shown in FIG. 7 , a significantdecrease in the expression amount of Snail1 and ZEB1 was recognized byreal-time PCR. Further, when the gene expression amount of anextracellular matrix-constituting protein in the iFCED was analyzed withSB431542 by real-time PCR, expression of collagen type I, collagen typeIV, collagen type VIII, and Fibronectin was significantly suppressed.

(Examination of the Expression of Collagen Type I, Collagen Type IV, andFibronectin by Immunostaining)

Then, expression of collagen type I, collagen type IV, and Fibronectinwas similarly examined by immunostaining. The immunostaining was carriedout in accordance with the above-described examples, provided that thefollowing antibodies were used as antibodies for collagen type I,collagen type IV, and Fibronectin.

-   -   An antibody against collagen type I: Anti collagen type I        (Rabbit polyclonal) (ROCKLLAND™ antibodies and assays, Cat no.:        600-401-103S)    -   An antibody against collagen type IV: collagen type IV (Rabbit        polyclonal)(Abcam, Cat no.: ab6586)    -   An antibody against Fibronectin: Anti-fibronectin (mouse        monoclonal) (BD Biosciences, Cat no.: 610077)

(Result)

The result is shown in FIG. 8 . As shown in FIG. 8 , it was confirmedthat TGF-beta signal inhibition using SB431542 also suppressedexpression of collagen type I, collagen type IV, collagen type VIII, andFibronectin in a protein level.

Example 6: An Effect of a TGF-Beta Signal Inhibiting Agent in the Caseof Fixing in a Confluent State One Week Later

In the present example, an effect of a TGF-beta signal inhibiting agentin the case of fixing in a confluent state one week later was confirmed.

Further, the iHCEC and the iFECD were cultured on Transwell PermeableSupports: 0.4 μm, six well plates (Costar, Cat no.: 3450) without serum,one week after which they were fixed in a confluent state andHE-stained. The HE-staining was carried out in accordance with theprocedure of the above-described examples.

(Result)

The result is shown in FIG. 9 . As shown in FIG. 9 , TGF-beta signalinhibition using SB431542 also suppressed overproduction of anextracellular matrix in the iFECD to result in the normal level.

The above description shows that a Fuchs' endothelial corneal dystrophypatient produces an excess amount of an extracellular matrix under aTGF-beta signal by promoting Snail1 or ZEB1 in comparison with a healthysubject. Further, it shows that suppression by siRNA or the like ofEMT-related genes such as Snail1, ZEB1, or the like and genes related toprotein production can suppress production of an extracellular matrix.In addition, it shows that inhibition of a TGF-beta signal can alsosuppress production of an extracellular matrix. It indicates thepossibility that suppression of inhibition of a TGF-beta signal,EMT-related genes such as Snail1, ZEB1, or the like, or a signal thereofcan suppress overproduction of an extracellular matrix of cornealendothelial cells of Fuchs' endothelial corneal dystrophy patients andsuppress guttae formation and thickening of Descemet's membrane.

As described above, the present invention is illustrated by preferableembodiments of the present invention. However, it will be understoodthat the scope of the present invention should be interpreted only bythe claims. It will be understood that the contents of patents, patentapplications, and literatures cited in the present specification shouldbe incorporated herein by reference as if their contents per se arespecifically described in the present specification.

INDUSTRIAL APPLICABILITY

Provided are techniques available in industries (the cell-culturingindustry, the pharmaceutical industry, and the like) relating totherapeutic or preventive medicaments for a disease, disorder, orcondition associated with extracellular matrix (ECM) abnormality in acorneal endothelium, in particular, extracellular matrix (ECM)abnormality in Fuchs' endothelial corneal dystrophy, and photophobia,wherein the medicaments comprises a TGF-beta signal inhibiting agent.

1. A medicament for treating or preventing a disease, disorder, orcondition associated with extracellular matrix (ECM) abnormality in acorneal endothelium, wherein the medicament comprises an effectiveamount of a TGF-beta signal inhibiting agent.
 2. The medicament fortreating or preventing according to claim 1, wherein the disease,disorder, or condition is a disorder related to Fuchs' endothelialcorneal dystrophy.
 3. The medicament for treating or preventingaccording to claim 1, wherein the disease, disorder, or conditioncomprises at least one selected from the group consisting ofphotophobia, blurred vision, vision disorder, eye pain, lacrimation,hyperemia, pain, bullous keratopathy, ophthalmic unpleasantness, adecrease in contrast, glare, edema in corneal stroma, bullouskeratopathy, and corneal opacity in Fuchs' endothelial cornealdystrophy.
 4. The medicament for treating or preventing according toclaim 1, wherein the TGF-beta signal inhibiting agent comprises at leastone of4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide,BMP-7, anti-TGF-beta antibody, anti-TGF-beta receptor antibody, siRNA ofTGF-beta, siRNA of a TGF-beta receptor, shRNA of TGF-beta, shRNA of aTGF-beta receptor, an aptamer of TGF-beta, an aptamer of a TGF-betareceptor, an antisense oligonucleotide of TGF-beta,6,7-dimethoxy-2-((2E)-3-(1-methyl-2-phenyl-1H-pyrrolo[2,3-b]pyridine-3-yl-prop-2-enoyl))-1,2,3,4-tetrahydroisoquinolone,3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide,2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,6-(4-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine,2-(5-chloro-2-fluorophenyl)-4-[(4-pyridinyl)amino]pteridine,4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, pharmaceuticallyacceptable salts or solvates thereof, or solvates of thepharmaceutically acceptable salts.
 5. The medicament for treating orpreventing according to claim 1, wherein the TGF-beta signal inhibitingagent comprises4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamideor a pharmaceutically acceptable salt thereof.
 6. The medicament fortreating or preventing according to claim 1, wherein the cornealendothelium is of a primate.
 7. The medicament for treating orpreventing according to claim 1, wherein the corneal endothelium is of ahuman.
 8. The medicament for treating or preventing according to claim1, comprising an additional medicinal component.
 9. The medicament fortreating or preventing according to claim 1, being eye-drops.